U.S. patent number 11,197,944 [Application Number 15/776,853] was granted by the patent office on 2021-12-14 for compositions and methods of mechanically inducing tissue regeneration.
This patent grant is currently assigned to CHARITE UNIVERSITATSMEDIZIN BERLIN, President and Fellows of Harvard College. The grantee listed for this patent is CHARITE--UNIVERSITAETSMEDIZIN BERLIN, President and Fellows of Harvard College. Invention is credited to Christine A. Cezar, Georg N. Duda, David J. Mooney, Ellen T. Roche, Herman H. Vandenburgh, Conor J. Walsh.
United States Patent |
11,197,944 |
Cezar , et al. |
December 14, 2021 |
Compositions and methods of mechanically inducing tissue
regeneration
Abstract
The present invention provides methods and compositions for
promoting regeneration of a tissue, methods for preventing or
reducing inflammation of a tissue, methods for preventing or
reducing fibrosis of a tissue, methods for increasing a mass of a
tissue, methods for increasing a level of oxygen available to a
tissue, methods for increasing a rate of metabolic waste removal
from a tissue, methods for increasing blood perfusion to a tissue,
and methods of treating severe muscle tissue damage in a subject in
need thereof by contacting the tissue with a composition that is
suitable for applying cyclic mechanical compression to the
tissue.
Inventors: |
Cezar; Christine A. (Cambridge,
MA), Walsh; Conor J. (Cambridge, MA), Mooney; David
J. (Sudbury, MA), Roche; Ellen T. (Galway,
IE), Vandenburgh; Herman H. (Providence, RI),
Duda; Georg N. (Berlin, DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College
CHARITE--UNIVERSITAETSMEDIZIN BERLIN |
Cambridge
Berlin |
MA
N/A |
US
DE |
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Assignee: |
President and Fellows of Harvard
College (Cambridge, MA)
CHARITE UNIVERSITATSMEDIZIN BERLIN (Berlin,
DE)
|
Family
ID: |
58719310 |
Appl.
No.: |
15/776,853 |
Filed: |
November 18, 2016 |
PCT
Filed: |
November 18, 2016 |
PCT No.: |
PCT/US2016/062685 |
371(c)(1),(2),(4) Date: |
May 17, 2018 |
PCT
Pub. No.: |
WO2017/087754 |
PCT
Pub. Date: |
May 26, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200222582 A1 |
Jul 16, 2020 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62305323 |
Mar 8, 2016 |
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62256877 |
Nov 18, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L
27/56 (20130101); A61L 27/14 (20130101); A61K
9/0024 (20130101); A61L 27/50 (20130101); A61K
47/36 (20130101); A61K 9/06 (20130101); A61L
27/042 (20130101); A61L 27/54 (20130101); A61K
35/12 (20130101); A61L 2430/30 (20130101); A61L
2400/06 (20130101) |
Current International
Class: |
A61L
27/04 (20060101); A61L 27/14 (20060101); A61L
27/56 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
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5098369 |
March 1992 |
Heilman et al. |
7361638 |
April 2008 |
Berlanga Acosta et al. |
7497837 |
March 2009 |
Sherman et al. |
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Foreign Patent Documents
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2011/075516 |
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Jun 2011 |
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WO |
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WO-2011075516 |
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Jun 2011 |
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WO |
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Primary Examiner: Song; Jianfeng
Attorney, Agent or Firm: McCarter & English, LLP
Zacharakis; Maria Laccotripe Song; Wei
Government Interests
GOVERNMENT SUPPORT
The invention was made with government support under DE013349
awarded by National Institutes of Health, DMR-0820484 awarded by
National Science of Foundation. The government has certain rights
in the invention.
Parent Case Text
RELATED APPLICATIONS
This application is a 35 U.S.C. .sctn. 371 national stage filing of
International Application No. PCT/US2016/062685, filed on Nov. 18,
2016, which in turn claims the benefit of priority to U.S.
Provisional Application No. 62/256,877, filed on Nov. 18, 2015 and
U.S. Provisional Application No. 62/305,323, filed on Mar. 8, 2016.
The entire contents of each of the foregoing patent applications
are incorporated herein by reference.
Claims
We claim:
1. A method for promoting regeneration of a tissue in a subject in
need thereof, comprising: contacting the tissue with a composition
comprising a matrix material and a magnetic material distributed
therethrough, wherein the composition comprises macropores having a
mean pore diameter in the range of about 10 .mu.m to about 10000
.mu.m, wherein the magnetic material is in the form of magnetic
particles having a size in the range from about 1 nm to about 500
nm, and wherein porosity, pore size, pore connectivity, and/or
specific volume of the composition changes by at least 10% in
response to an electromagnetic signal; and applying cyclic
mechanical compressions to the tissue, thereby promoting
regeneration of the tissue, wherein the composition is free of a
bioactive agent, a therapeutic agent, a cell, or a combination
thereof.
2. The method of claim 1, wherein the cyclic mechanical
compressions are caused by the electromagnetic signal, or by
pneumatic or hydraulic actuation.
3. The method of claim 1, wherein the composition comprises a
swelling agent, wherein the swelling agent concentration changes by
at least 10% in response to an electromagnetic signal.
4. The method of claim 1, wherein the composition has a porosity of
0.1 to 0.99.
5. The method of claim 1, wherein the matrix material is a polymer,
a cross-linked polymer, a copolymer, or a block polymer gel.
6. The method of claim 1, wherein the matrix material comprises a
polymer selected from the group consisting of polyurethanes,
glycosaminoglycan, silk, fibrin, poly-ethyleneglycol (PEG),
polyhydroxy ethyl methacrylate, polyvinyl alcohol, polyacrylamide,
poly (N-vinyl pyrolidone), poly(lactic acid), poly glycolic acid
(PGA), poly lactic-co-glycolic acid (PLGA), poly e-carpolactone
(PCL), polyethylene oxide, poly propylene fumarate (PPF), poly
acrylic acid (PAA), polyhydroxybutyric acid, hydrolysed
polyacrylonitrile, polymethacrylic acid, polyethylene amine, esters
of alginic acid; pectinic acid; alginate, fully or partially
oxidized alginate, hyaluronic acid, carboxy methyl cellulose,
heparin, heparin sulfate, chitosan, carboxymethyl chitosan, chitin,
pullulan, gellan, xanthan, collagen, gelatin, carboxymethyl starch,
carboxymethyl dextran, chondroitin sulfate, cationic guar, cationic
starch, and combinations thereof.
7. The method of claim 1, wherein the magnetic material is
ferromagnetic, ferrimagnetic, diamagnetic, paramagnetic, or
superparamagnetic material.
8. The method of claim 1, wherein the macropores have a mean pore
diameter in the range of about 150 .mu.m to about 7500 .mu.m.
9. The method of claim 1, wherein porosity, pore size, pore
connectivity, swelling agent concentration, and/or specific volume
changes by at least 25% in response to the electromagnetic
signal.
10. The method of claim 1, wherein the magnetic material is
distributed homogeneously within the matrix material.
11. The method of claim 1, wherein the magnetic material is
distributed heterogeneously within the matrix material.
12. The method of claim 11, wherein the heterogeneous distribution
of the magnetic material within the matrix material is formed by
application of a magnetic field during polymerization of the matrix
material.
13. The method of claim 11, wherein the magnetic material is
distributed into a separate compartment within the matrix material
or is distributed at one side within the matrix material distant
from the electromagnetic signal.
14. The method of claim 1, wherein the composition is suitable for
implantation within the tissue.
15. The method of claim 1, wherein the tissue is damaged and the
cyclic mechanical compressions are applied to the site of tissue
damage (i) within less than 5, 10, 20, 30, 40, 50, 60 minutes after
the damage has occurred; (ii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 12, or 24 hours after the damage has occurred; (iii) at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10 11, 12, 24, 48 or 60 months after the damage has occurred;
(iv) over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 24, 36, 48, 60, 72, 84, 96 or 120 hours; (v) over a period of
at least 14 days; or (vi) for about 1 to 30 days, about 1 to 50
days, about 1 to 100 days, about 1 to 200 days or about 1 to 300
days.
Description
BACKGROUND OF THE INVENTION
Skeletal muscle comprises a large percentage of the human body mass
(40-50%) and plays an essential role in locomotion, postural
support, and breathing. In response to minor injuries, skeletal
muscle possesses a remarkable capacity for regeneration. Small
exercise-induced tears, lacerations, and contusions typically heal
without therapeutic intervention (Juhas M & Bursac N (2013)
Curr. Opin. Biotechnol. 24(5):880-886; Turner N J & Badylak S F
(2012) Cell Tissue Res. 347(3):759-774). However, severe injuries
resulting in a muscle mass loss of greater than 20% can lead to
extensive fibrosis and loss of muscle function (Turner N J &
Badylak S F (2012) Cell Tissue Res. 347(3):759-774). Unfortunately,
traumatic injuries resulting from motor vehicle accidents,
aggressive tumor ablation, and prolonged denervation are common
clinical situations and frequently lead to volumetric muscle loss
(Turner N J & Badylak S F (2012) Cell Tissue Res.
347(3):759-774; Jarvinen TAHJ, et al. (2005) Am. J. Sports Med.
33(5):745-764; Rossi C A, et al. (2010) Organogenesis
6(3):167-172). While surgical reconstruction can lead to improved
outcomes, this technique typically does not fully regenerate lost
muscle tissue and often leads to donor site morbidity (Ma C H, et
al. (2008) Injury 39 Suppl 4:67-74; Tu Y K, et al. (2008) Injury 39
Suppl 4:75-95). As a result, the development of therapeutic
strategies to treat severe skeletal muscle injuries is an area of
active investigation.
Typical current approaches to skeletal muscle repair rely upon the
delivery of biologics such as growth factors and cells to enhance
muscle regeneration. In early clinical trials, the intramuscular
injection of cultured myoblasts was proven to be a safe but
ineffective cell therapy for human myopathies, likely due to rapid
death, poor migration, and immune rejection of the donor cells
(Tedesco F S & Cossu G (2012) Curr. Opin. Neurol.
25(5):597-603; Palmieri B, et al. (2010) Pediatr. Transplant.
14(7):813-819). Recently, the identification of several important
microenvironmental cues that regulate satellite cell fate has led
to the development of cell-instructive biomaterials that improve
cell engraftment and muscle regeneration. Biomaterial-based
delivery of myogenic (IGF, FGF-2, HGF) and angiogenic factors
(VEGF) that appear during the normal regenerative process has
proven successful in animal models of severe muscle injury (Ten
Broek R W, et al. (2010) J. Cell. Physiol. 224(1):7-16; Shansky J,
et al. (2006) Tissue Eng. 12(7):1833-1841; Pelosi L, et al. (2007)
FASEB J. 21(7):1393-1402; Silva E A & Mooney D J (2007) J.
Thromb. Haemost. 5(3):590-598; Borselli C, et al. (2010) Proc.
Natl. Acad. Sci. U.S.A. 107(8):3287-3292). Furthermore, the
synergistic presentation of cells and growth factors that mimic
normal in vivo presentation has led to improved functional muscle
regeneration in mice (Borselli C, et al. (2010) Proc. Natl. Acad.
Sci. U.S.A. 107(8):3287-3292; Sheehan S M & Allen R E (1999) J.
Cell. Physiol. 181(3):499-506). However, although much progress has
been made toward the development of cell and growth factor-based
approaches for the treatment of severely injured skeletal muscle in
rodents, reliable clinical therapies still do not exist.
Accordingly, there remains an ongoing and unmet need for the
development of novel therapeutic strategies to treat severe muscle
injuries.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery
that devices with a capacity to apply cyclic compression forces
such as biphasic ferrogels and pressure cuffs can be used to
mechanically stimulate and regenerate injured tissue, e.g., muscle
tissue, without the use of growth factors or cells. In particular,
both magnetic actuation of biphasic ferrogel scaffolds implanted at
the site of muscle injury and external actuation of compression
device surrounding the site of muscle injury resulted in uniform
cyclic compressions that led to reduced fibrous capsule formation
around the implant, as well as reduced fibrosis and inflammation in
the injured muscle. Furthermore, ferrogel-driven and pressure
cuff-driven mechanical compressions led to enhanced muscle
regeneration and an approximately 3 and 2.2-fold increase in
maximum contractile force of the treated muscle, respectively.
Without intending to be limited by theory, it is believed that
these biologic-free devices exhibit a potential immunomodulatory
role when stimulated and could potentially translate rapidly to the
clinic. In addition, the therapeutic use of direct mechanical
stimulation of injured tissues via externally actuated compression
devices could establish a new paradigm for regenerative
medicine.
Accordingly, in one aspect, the present invention provides methods
for promoting regeneration of a tissue in a subject in need
thereof. The methods include contacting the tissue with a
composition comprising a matrix material and a magnetic material
distributed therethrough, wherein the composition comprises
macropores having a mean pore diameter in the range of about 10
.mu.m to about 10000 .mu.m, e.g., about 10 .mu.m to 1000 .mu.m,
about 50 .mu.m to 1000 .mu.m, about 100 .mu.m to 1000 .mu.m, about
150 .mu.m to 1000 .mu.m, about 200 .mu.m to 1000 .mu.m, about 300
.mu.m to 1000 .mu.m, about 400 .mu.m to 1000 .mu.m, about 500 .mu.m
to 1000 .mu.m, about 600 .mu.m to 1000 .mu.m, about 1000 .mu.m to
10000 .mu.m, about 2000 .mu.m to 10000 .mu.m, about 3000 .mu.m to
10000 .mu.m, about 4000 .mu.m to 10000 .mu.m, about 5000 .mu.m to
10000 .mu.m, or about 6000 .mu.m to 10000 .mu.m, wherein the
magnetic material is in the form of magnetic particles having a
size in the range from about 1 nm to about 500 nm, and wherein
porosity, pore size, pore connectivity, swelling agent
concentration and/or specific volume of the composition changes by
at least 10% in response to an electromagnetic signal; and applying
cyclic mechanical compressions to the tissue, thereby promoting
regeneration of the tissue.
In some embodiments, the cyclic mechanical compressions are caused
by an electromagnetic signal. In other embodiments, the cyclic
mechanical compressions are caused by pneumatic or hydraulic
actuation.
In some embodiments, the composition comprises a swelling agent. In
other embodiments, the composition has a porosity of 0.1 to
0.99.
In some embodiments, the matrix material is a polymer, a copolymer,
or a block polymer gel. In other embodiments, the matrix material
is a cross-linked polymer, a copolymer, or a block polymer gel. In
some embodiments, the matrix material comprises a polymer selected
from the group consisting of polyurethanes, glycosaminoglycan,
silk, fibrin, MATRIGEL.RTM., poly-ethyleneglycol (PEG), polyhydroxy
ethyl methacrylate, polyvinyl alcohol, polyacrylamide, poly
(N-vinyl pyrolidone), poly(lactic acid), poly glycolic acid (PGA),
poly lactic-co-glycolic acid (PLGA), poly e-carpolactone (PCL),
polyethylene oxide, poly propylene fumarate (PPF), poly acrylic
acid (PAA), polyhydroxybutyric acid, hydrolysed polyacrylonitrile,
polymethacrylic acid, polyethylene amine, esters of alginic acid;
pectinic acid; and alginate, fully or partially oxidized alginate,
hyaluronic acid, carboxy methyl cellulose, heparin, heparin
sulfate, chitosan, carboxymethyl chitosan, chitin, pullulan,
gellan, xanthan, collagen, gelatin, carboxymethyl starch,
carboxymethyl dextran, chondroitin sulfate, cationic guar, cationic
starch, and combinations thereof.
In some embodiments, the magnetic material is ferromagnetic,
ferrimagnetic, diamagnetic, paramagnetic, or superparamagnetic
material. In other embodiments, the magnetic material is an iron
oxide particle. In some embodiments, the iron oxide particle is
magnetite. In other embodiments, the iron oxide particle is
maghemite.
In some embodiments, the macropores have a mean pore diameter in
the range of about 150 .mu.m to about 7500 .mu.m, e.g., about 150
.mu.m to 1000 .mu.m, about 150 .mu.m to 2000 .mu.m, about 150 .mu.m
to 3000 .mu.m, about 150 .mu.m to 4000 .mu.m, about 150 .mu.m to
5000 .mu.m, about 150 .mu.m to 6000 .mu.m, about 150 .mu.m to 7000
.mu.m, about 500 .mu.m to 1000 .mu.m, about 600 .mu.m to 1000
.mu.m, about 1000 .mu.m to 2000 .mu.m, 2000 .mu.m to 4000 .mu.m,
3000 .mu.m to 5000 .mu.m, 4000 .mu.m to 6000 .mu.m, or about 5000
.mu.m to 7000 .mu.m.
In some embodiments, the composition comprises a bioactive agent.
In other embodiments, the bioactive agent is covalently linked to
the matrix material. In some embodiments, the bioactive agent is a
therapeutic agent.
In some embodiments, the composition comprises a cell. In other
embodiments, the composition is free of a bioactive agent or a
cell.
In some embodiments, porosity, pore size, pore connectivity,
swelling agent concentration, and/or specific volume changes by at
least 25% in response to the electromagnetic signal. In other
embodiments, porosity, pore size, pore connectivity, swelling agent
concentration, and/or specific volume increases in response to the
electromagnetic signal. In yet another embodiment, porosity, pore
size, pore connectivity, swelling agent concentration, and/or
specific volume decreases in response to the electromagnetic
signal.
In some embodiments, the electromagnetic signal is generated by
application of a magnetic field.
In some embodiments, the magnetic material is distributed
homogeneously within the matrix material. In other embodiments, the
magnetic material is distributed heterogeneously within the matrix
material. In some embodiments, the heterogeneous distribution of
the magnetic material within the matrix material is formed by
application of a magnetic field during polymerization of the matrix
material. In other embodiments, the magnetic material is
distributed into a separate compartment within the matrix material.
In yet another embodiment, the magnetic material is distributed at
one side within the matrix material distant from the
electromagnetic signal.
In some embodiments, the composition is suitable for implantation
within the tissue. In some embodiments, the tissue is selected from
the group consisting of a muscle tissue, a heart tissue, a blood
vessel tissue, a skin tissue, a bone tissue, a cartilage tissue, a
connective tissue, a tendon tissue, and a ligament tissue.
In some embodiments, the tissue is a muscle tissue. In other
embodiments, the muscle tissue is selected from the group
consisting of a skeletal muscle tissue, a smooth muscle tissue and
a cardiac muscle tissue.
In some embodiments, the muscle tissue in the subject is damaged.
In other embodiments, the muscle tissue damage is induced by
exercise. In some embodiments, the muscle tissue damage is induced
by a myotoxin. In other embodiments, the muscle tissue damage is
induced by ischemia. In some embodiments, the muscle tissue damage
is induced by hind limb ischemia. In other embodiments, the muscle
tissue damage is induced by a physical trauma. In some embodiments,
the muscle tissue damage is induced by cryo-damages. In other
embodiments, the muscle tissue damage is induced by muscle
degeneration. In some embodiments, the muscle tissue damage is
induced by age-related muscle loss. In other embodiments, the
muscle tissue damage results in a muscle mass loss or injury of
about 0.01% to 99.9%, e.g., a muscle mass loss or injury of greater
than 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, or 90%. In some embodiments, the muscle tissue
damage results in a muscle mass loss or injury of about greater
than 20%.
In some embodiments, the composition is implanted at the site of
tissue damage. In other embodiments, the cyclic mechanical
compressions are applied to the site of tissue damage after the
damage has occurred.
In some embodiments, the cyclic mechanical compressions are applied
to the site of tissue damage immediately after the damage has
occurred, or within less than 5, 10, 20, 30, 40, 50, 60 minutes
after the damage has occurred. In other embodiments, the cyclic
mechanical compressions are applied to the site of tissue damage at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 24 hours after the
damage has occurred. In some embodiments, the cyclic mechanical
compressions are applied to the site of tissue damage at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 days or at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 11, 12, 24, 48 or 60 months after the damage has
occurred.
In some embodiments, the cyclic mechanical compressions are applied
to the site of tissue damage over a period of time. In other
embodiments, the cyclic mechanical compressions are applied to the
site of tissue damage for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 24, 36, 48, 60, 72, 84, 96 or 120 hours. In some
embodiments, the cyclic mechanical compressions are applied at
least once daily.
In some embodiments, the cyclic mechanical compressions are applied
to the site of tissue damage for about 1 to 30 days, about 1 to 50
days, about 1 to 100 days, about 1 to 200 days or about 1 to 300
days. In other embodiments, the cyclic mechanical compressions are
applied to the site of tissue damage over a period of at least 14
days, e.g., at least 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 days.
In some embodiments, the methods further comprises determining the
level of a cytokine in a subject.
In some embodiments, the subject is a mammal. In other embodiments,
the subject is a mammal selected from the group consisting of a
human, a horse, a dog, a cat, a cow, a mouse, a rabbit, and a
rat.
In one aspect, the present invention provides methods of increasing
a mass of a tissue in a subject in need thereof. The methods
include contacting the tissue with a composition comprising a
matrix material and a magnetic material distributed therethrough,
wherein the composition comprises macropores having a mean pore
diameter in the range of about 10 .mu.m to about 10000 .mu.m,
wherein the magnetic material is in the form of magnetic particles
having a size in the range from about 1 nm to about 500 nm, and
wherein porosity, pore size, pore connectivity, swelling agent
concentration and/or specific volume of the composition changes by
at least 10% in response to an electromagnetic signal; and applying
cyclic mechanical compressions to the tissue, thereby increasing
the mass of the tissue.
In some embodiments, the cyclic mechanical compressions are caused
by an electromagnetic signal. In other embodiments, the cyclic
mechanical compressions are caused by pneumatic or hydraulic
actuation.
In some embodiments, the tissue is a muscle tissue.
In another aspect, the present invention provides methods of
enhancing a function of a tissue in a subject in need thereof. The
methods include contacting the tissue with a composition comprising
a matrix material and a magnetic material distributed therethrough,
wherein the composition comprises macropores having a mean pore
diameter in the range of about 10 .mu.m to about 10000 .mu.m,
wherein the magnetic material is in the form of magnetic particles
having a size in the range from about 1 nm to about 500 nm, and
wherein porosity, pore size, pore connectivity, swelling agent
concentration and/or specific volume of the composition changes by
at least 10% in response to an electromagnetic signal; and applying
cyclic mechanical compressions to the tissue, thereby enhancing the
function of the tissue.
In some embodiments, the cyclic mechanical compressions are caused
by the electromagnetic signal. In other embodiments, the cyclic
mechanical compressions are caused by pneumatic or hydraulic
actuation.
In some embodiments, the tissue is a muscle tissue. In other
embodiments, the contractile force of the tissue is increased.
In one aspect, the present invention provides methods for
preventing or reducing inflammation of a tissue in a subject in
need thereof. The methods include contacting the tissue with a
composition comprising a matrix material and a magnetic material
distributed therethrough, wherein the composition comprises
macropores having a mean pore diameter in the range of about 10
.mu.m to about 10000 .mu.m, wherein the magnetic material is in the
form of magnetic particles having a size in the range from about 1
nm to about 500 nm, and wherein porosity, pore size, pore
connectivity, swelling agent concentration and/or specific volume
of the composition changes by at least 10% in response to an
electromagnetic signal; and applying cyclic mechanical compressions
to the tissue, thereby preventing or reducing inflammation of the
tissue.
In some embodiments, the cyclic mechanical compressions are caused
by an electromagnetic signal. In other embodiments, the cyclic
mechanical compressions are caused by pneumatic or hydraulic
actuation.
In some embodiments, the tissue is damaged in the subject. In some
embodiments, the tissue is a muscle tissue.
In some embodiments, the composition is implanted at a site of
tissue damage. In other embodiments, fibrosis at the site of tissue
damage is reduced. In some embodiments, inflammatory cell removal
at the site of tissue damage is accelerated.
In some embodiments, the methods comprise determine the level of a
cytokine in a subject is increased. In other embodiments, the level
of a cytokine in a subject is reduced. In some embodiments, the
level of a pro-inflammatory cytokine in a subject is reduced. In
other embodiments, the pro-inflammatory cytokine is selected from
the group consisting of myeloperoxidase, neutrophil
gelatinase-associated lipocalin, interleukin-17A and interleukin-6.
In other embodiments, the level of a pro-inflammatory cytokine in a
subject is reduced by enhanced intramuscular convection driven by
cyclic mechanical compressions of the muscle tissue.
In another aspect, the present invention provides methods of
preventing or reducing fibrosis of a tissue in a subject in need
thereof. The methods include contacting the tissue with a
composition comprising a matrix material and a magnetic material
distributed therethrough, wherein the composition comprises
macropores having a mean pore diameter in the range of about 10
.mu.m to about 10000 .mu.m, wherein the magnetic material is in the
form of magnetic particles having a size in the range from about 1
nm to about 500 nm, and wherein porosity, pore size, pore
connectivity, swelling agent concentration and/or specific volume
of the composition changes by promoting at least 10% in response to
an electromagnetic signal; and applying cyclic mechanical
compressions to the tissue, thereby preventing or reducing fibrosis
of the tissue.
In some embodiments, the cyclic mechanical compressions are caused
by an electromagnetic signal. In other embodiments, the cyclic
mechanical compressions are caused by pneumatic or hydraulic
actuation.
In some embodiments, the tissue is damaged. In some embodiments,
the tissue is a muscle tissue.
In some embodiments, the composition is implanted at a site of
tissue damage. In other embodiments, the formation of a fibrous
capsule at the site of tissue damage is reduced. In some
embodiments, the thickness of the fibrous capsule at the site of
tissue damage is reduced. In other embodiments, inflammatory cell
removal at the site of tissue damage is accelerated.
In one aspect, the present invention provides methods of increasing
a level of oxygen available to a tissue in a subject in need
thereof. The methods include contacting the tissue with a
composition comprising a matrix material and a magnetic material
distributed therethrough, wherein the composition comprises
macropores having a mean pore diameter in the range of about 10
.mu.m to about 10000 .mu.m, wherein the magnetic material is in the
form of magnetic particles having a size in the range from about 1
nm to about 500 nm, and wherein porosity, pore size, pore
connectivity, swelling agent concentration and/or specific volume
of the composition changes by promoting at least 10% in response to
an electromagnetic signal; and applying cyclic mechanical
compressions to the tissue, thereby increasing the level of oxygen
available to the tissue.
In some embodiments, the cyclic mechanical compressions are caused
by an electromagnetic signal. In other embodiments, the cyclic
mechanical compressions are caused by pneumatic or hydraulic
actuation.
In some embodiments, the tissue is a muscle tissue. In some
embodiments, the oxygen level is increased by increasing blood flow
to the muscle tissue. In other embodiments, the oxygen level is
increased by enhanced intramuscular convection driven by cyclic
mechanical compressions of the muscle tissue.
In another aspect, the present invention provides methods of
increasing a rate of metabolic waste product removal from a tissue
in a subject in need thereof. The methods include contacting the
tissue with a composition comprising a matrix material and a
magnetic material distributed therethrough, wherein the composition
comprises macropores having a mean pore diameter in the range of
about 10 .mu.m to about 10000 .mu.m, wherein the magnetic material
is in the form of magnetic particles having a size in the range
from about 1 nm to about 500 nm, and wherein porosity, pore size,
pore connectivity, swelling agent concentration and/or specific
volume of the composition changes by promoting at least 10% in
response to an electromagnetic signal; and applying cyclic
mechanical compressions to the tissue, thereby increasing the rate
of metabolic waste product removal from the tissue.
In some embodiments, the cyclic mechanical compressions are caused
by an electromagnetic signal. In other embodiments, the cyclic
mechanical compressions are caused by pneumatic or hydraulic
actuation.
In some embodiments, the tissue is a muscle tissue. In some
embodiments, the rate of metabolic waste product removal is
increased by enhanced fluid transportation driven by cyclic
mechanical compressions around the tissue.
In one aspect, the present invention provides methods of increasing
blood perfusion to a tissue in a subject in need thereof. The
methods include contacting the tissue with a composition comprising
a matrix material and a magnetic material distributed therethrough,
wherein the composition comprises macropores having a mean pore
diameter in the range of about 10 .mu.m to about 10000 .mu.m,
wherein the magnetic material is in the form of magnetic particles
having a size in the range from about 1 nm to about 500 nm, and
wherein porosity, pore size, pore connectivity, swelling agent
concentration and/or specific volume of the composition changes by
promoting at least 10% in response to an electromagnetic signal;
and applying cyclic mechanical compressions to the tissue, thereby
increasing blood perfusion to the tissue.
In some embodiments, the cyclic mechanical compressions are caused
by an electromagnetic signal. In other embodiments, the cyclic
mechanical compressions are caused by pneumatic or hydraulic
actuation.
In some embodiments, the tissue is a muscle tissue.
In one aspect, the present invention provides methods of treating a
severe muscle tissue damage in a subject in need thereof. The
methods include contacting the muscle tissue with a composition
comprising a matrix material and a magnetic material distributed
therethrough, wherein the composition comprises macropores having a
mean pore diameter in the range of about 10 .mu.m to about 10000
.mu.m, wherein the magnetic material is in the form of magnetic
particles having a size in the range from about 1 nm to about 500
nm, and wherein porosity, pore size, pore connectivity, swelling
agent concentration and/or specific volume of the composition
changes by promoting at least 10% in response to an electromagnetic
signal; and applying cyclic mechanical compressions to the muscle
tissue, thereby treating the severe muscle tissue damage in the
subject.
In some embodiments, the cyclic mechanical compressions are caused
by an electromagnetic signal. In other embodiments, the cyclic
mechanical compressions are caused by pneumatic or hydraulic
actuation.
In one aspect, the present invention provides methods for promoting
regeneration of a tissue in a subject in need thereof. The methods
include contacting the tissue with a compression device suitable
for applying cyclic mechanical compressions at a site of tissue
damage, and applying cyclic mechanical compressions to the tissue
using the compression device, thereby promoting regeneration of the
tissue at the site of tissue damage.
In some embodiments, the site of tissue damage is on or in a limb,
a spine, a neck, a waist, a shoulder, a knee, or a joint of the
subject. In some embodiments, the limb is a lower limb of the
subject and the site of the tissue damage is on or in an ankle, a
calf, a thigh, or a foot. In other embodiments, the limb is a upper
limb of the subject is and the site of the tissue damage is on or
in a hand, a wrist, an arm, a shoulder or an axilla.
In some embodiments, the compression device is electromagnetically
actuated, pneumatically actuated or hydraulically actuated to apply
the cyclic mechanical compressions to the tissue.
In some embodiments, the compression device comprises a surrounding
member configured to surround the site of tissue damage and apply
compression to the site of the tissue damage. In some embodiments,
contacting the tissue with the compression device suitable for
applying cyclic mechanical compressions at the site of tissue
damage comprises disposing the surrounding member encircling a body
part that includes the site of tissue damage. In some embodiments,
the surrounding member is disposed externally to the body and
encircling the body part. In other embodiments, the surrounding
member is disposed at least partially internally within the body
and encircling the body part.
In some embodiments, applying cyclic mechanical compressions to the
tissue using the compression device comprises using a controller
associated with the surrounding member to generate the cyclic
mechanical compressions in the surrounding member.
In some embodiments, the compression device further comprises the
controller. In some embodiments, the controller is programmable to
achieve different motions and forces; and wherein the method
further comprises selecting a desired motion or a desired force
using the controller prior to or during applying cyclic mechanical
compressions to the tissue using the compression device. In some
embodiments, the desired motion comprises one or more of bending,
twisting or squeezing.
In some embodiments, the surrounding member comprises a soft
actuator; and wherein the cyclic mechanical compressions are
applied to the tissue, at least in part, by actuation of the soft
actuator. In other embodiments, the surrounding member further
comprises an inflatable portion having a channel; and wherein the
soft actuator is disposed in the channel.
In some embodiments, the soft actuator is selected from the group
consisting of a fiber reinforced actuator, a Pneunet bending
actuator, a McKibben actuator, a pleated air muscle, a balloon, an
inflatable device, a motor, a vibrating motor, a cable, an
electroactive material, e.g., a shape memory alloy, an
electrostatic, a dielectric elastomer, and combinations
thereof.
In some embodiments, the surrounding member comprises one or more
inflatable portions. In other embodiments, the one or more
inflatable portions comprise one or more inflatable members. In
some embodiments, the one or more inflatable members comprise a
balloon; and wherein the surrounding member further comprises a
sleeve comprising a textile, a mesh, a fabric, a silicone elastomer
or a rubber configured to hold the balloon against a site of tissue
damage.
In some embodiments, applying cyclic mechanical compressions to the
tissue using the compression device comprises adjusting an
inflation pressure of at least some of the one or more inflatable
portions using a controller associated with the surrounding
member.
In some embodiments, contacting the tissue with a compression
device suitable for applying cycle mechanical compressions at the
site of tissue damage comprises at least partially inflating at
least some of the one or more inflatable portions or increasing an
inflation pressure applied to the one or more inflatable
portions.
In some embodiments, the one or more inflatable portions comprise a
plurality of independently inflatable portions. In some
embodiments, applying cyclic mechanical compressions to the tissue
using the compression device comprises individually controlling an
inflation pressure of each of the independently inflatable portions
using the controller associated with the surrounding member. In
other embodiments, the cyclic mechanical compressions are applied
to only a portion of the tissue underlying the surrounding member,
wherein the portion of the tissue is at, overlies or is adjacent to
the site of tissue damage.
In some embodiments, the compression device is configured to be
customizable for different geometries and morphologies. In other
embodiments, the compression device is suitable for wearable
applications; and wherein the subject is wearing the compression
device while the cyclic mechanical compressions are applied to the
tissue.
In some embodiments, the compression device is suitable for
generating active cyclic mechanical compressions.
In some embodiments, the composition is suitable for implantation
within the tissue. In some embodiments, the tissue is selected from
the group consisting of a muscle tissue, a heart tissue, a blood
vessel tissue, a skin tissue, a bone tissue, a cartilage tissue, a
connective tissue, a tendon tissue, and a ligament tissue.
In some embodiments, the tissue is a muscle tissue. In other
embodiments, the muscle tissue is selected from the group
consisting of a skeletal muscle tissue, a smooth muscle tissue and
a cardiac muscle tissue.
In some embodiments, the muscle tissue in the subject is damaged.
In other embodiments, the muscle tissue damage is induced by
exercise. In some embodiments, the muscle tissue damage is induced
by a myotoxin. In other embodiments, the muscle tissue damage is
induced by ischemia. In some embodiments, the muscle tissue damage
is induced by hind limb ischemia. In other embodiments, the muscle
tissue damage is induced by a physical trauma. In some embodiments,
the muscle tissue damage is induced by cryo-damages. In other
embodiments, the muscle tissue damage is induced by muscle
degeneration. In some embodiments, the muscle tissue damage is
induced by age-related muscle loss. In other embodiment, the muscle
tissue damage results in a muscle mass loss or injury of about
0.01% to 99.9%, e.g., a muscle mass loss or injury of greater than
0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, or 90%. In some embodiments, the muscle tissue damage
results in a muscle mass loss or injury of about greater than
20%.
In some embodiments, the composition is implanted at the site of
tissue damage. In other embodiments, the cyclic mechanical
compressions are applied to the site of tissue damage after the
damage has occurred.
In some embodiments, the cyclic mechanical compressions are applied
to the site of tissue damage immediately after the damage has
occurred, or within less than 5, 10, 20, 30, 40, 50, 60 minutes
after the damage has occurred. In other embodiments, the cyclic
mechanical compressions are applied to the site of tissue damage at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 24 hours after the
damage has occurred. In some embodiments, the cyclic mechanical
compressions are applied to the site of tissue damage at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 days or at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 11, 12, 24, 48 or 60 months after the damage has
occurred.
In some embodiments, the cyclic mechanical compressions are applied
to the site of tissue damage over a period of time. In other
embodiments, the cyclic mechanical compressions are applied to the
site of tissue damage for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 24, 36, 48, 60, 72, 84, 96 or 120 hours. In some
embodiments, the cyclic mechanical compressions are applied at
least once daily.
In some embodiments, the cyclic mechanical compressions are applied
to the site of tissue damage for about 1 to 30 days, about 1 to 50
days, about 1 to 100 days, about 1 to 200 days or about 1 to 300
days. In other embodiments, the cyclic mechanical compressions are
applied to the site of tissue damage over a period of at least 14
days, e.g., at least 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 days.
In some embodiments, the compression device is configured to exert
a peak pressure of about 0.1 kPa to 1000 kPa, about 1-10 kPa, about
1-20 kPa, about 1-30 kPa, about 1-50 kPa, about 1-100 kPa, about
1-1000 kPa, about 10-100 kPa, about 10-200 kPa, about 10-300 kPa,
about 10-500 kPa, about 10-1000 kPa, about 100-1000 kPa, about
200-1000 kPa, about 300-1000 kPa, about 400-1000 kPa, about
500-1000 kPa, about 600-1000 kPa, about 700-1000 kPa, about
800-1000 kPa, or about 900-1000 kPa.
In some embodiments, the subject is a mammal. In other embodiments,
the subject is a mammal selected from the group consisting of a
human, a horse, a dog, a cat, a cow, a mouse, a rabbit, and a
rat.
In one aspect, the present invention provides methods for
preventing or reducing inflammation of a tissue in a subject in
need thereof. The methods include contacting the tissue with a
compression device suitable for applying cyclic mechanical
compressions at a site of tissue damage, and applying cyclic
mechanical compressions to the tissue using the compression device
using the compression device, thereby preventing or reducing
inflammation of the tissue.
In some embodiments, the tissue is a muscle tissue. In other
embodiments, fibrosis at the site of tissue damage is reduced. In
some embodiments, inflammatory cell removal at the site of tissue
damage is accelerated.
In some embodiments, the methods comprise determine the level of a
cytokine in a subject is increased. In other embodiments, the level
of a cytokine in a subject is reduced. In some embodiments, the
level of a pro-inflammatory cytokine in a subject is reduced. In
other embodiments, the pro-inflammatory cytokine is selected from
the group consisting of myeloperoxidase, neutrophil
gelatinase-associated lipocalin, interleukin-17A and interleukin-6.
In other embodiments, the level of a pro-inflammatory cytokine in a
subject is reduced by enhanced intramuscular convection driven by
cyclic mechanical compressions of the muscle tissue.
In another aspect, the present invention provides methods of
preventing or reducing fibrosis of a tissue in a subject in need
thereof. The methods include contacting the tissue with a
compression device suitable for applying cyclic mechanical
compressions at a site of tissue damage, and applying cyclic
mechanical compressions to the tissue using the compression device
using the compression device, thereby preventing or reducing
fibrosis of the tissue.
In some embodiments, the tissue is a muscle tissue. In other
embodiments, the formation of a fibrous capsule at the site of
tissue damage is reduced. In some embodiments, the thickness of the
fibrous capsule at the site of tissue damage is reduced. In other
embodiments, inflammatory cell removal at the site of tissue damage
is accelerated.
In one aspect, the present invention provides methods of increasing
a mass of a tissue in a subject in need thereof. The methods
include contacting the tissue with a compression device suitable
for applying cyclic mechanical compressions at a site of tissue
damage, and applying cyclic mechanical compressions to the tissue
using the compression device using the compression device, thereby
increasing the mass of the tissue.
In some embodiments, the tissue is a muscle tissue.
In another aspect, the present invention provides methods of
enhancing a function of a tissue in a subject in need thereof. The
methods include contacting the tissue with a compression device
suitable for applying cyclic mechanical compressions at a site of
tissue damage, and applying cyclic mechanical compressions to the
tissue using the compression device using the compression device,
thereby enhancing the function of the tissue.
In some embodiments, the tissue is a muscle tissue. In other
embodiments, the contractile force of the tissue is increased.
In one aspect, the present invention provides methods of increasing
a level of oxygen available to a tissue in a subject in need
thereof. The methods include contacting the tissue with a
compression device suitable for applying cyclic mechanical
compressions at a site of tissue damage, and applying cyclic
mechanical compressions to the tissue using the compression device
using the compression device, thereby increasing the level of
oxygen available to the tissue.
In some embodiments, the tissue is a muscle tissue. In some
embodiments, the oxygen level is increased by increasing blood flow
to the muscle tissue. In other embodiments, the oxygen level is
increased by enhanced intramuscular convection driven by cyclic
mechanical compressions of the muscle tissue.
In another aspect, the present invention provides methods of
increasing a rate of metabolic waste product removal from a tissue
in a subject in need thereof. The methods include contacting the
tissue with a compression device suitable for applying cyclic
mechanical compressions at a site of tissue damage, and applying
cyclic mechanical compressions to the tissue using the compression
device using the compression device, thereby increasing the rate of
metabolic waste product removal from the tissue.
In some embodiments, the tissue is a muscle tissue. In some
embodiments, the rate of metabolic waste product removal is
increased by enhanced fluid transportation driven by cyclic
mechanical compressions around the tissue.
In one aspect, the present invention provides methods of increasing
blood perfusion to a tissue in a subject in need thereof. The
methods include contacting the tissue with a compression device
suitable for applying cyclic mechanical compressions at a site of
tissue damage, and applying cyclic mechanical compressions to the
tissue using the compression device using the compression device,
thereby increasing blood perfusion to the tissue.
In some embodiments, the tissue is a muscle tissue.
In one aspect, the present invention provides methods of treating a
severe muscle tissue damage in a subject in need thereof. The
methods include contacting the muscle tissue with a compression
device suitable for applying cyclic mechanical compressions at a
site of muscle tissue damage, and applying cyclic mechanical
compressions to the muscle tissue using the compression device
using the compression device, thereby treating a severe muscle
tissue damage in the subject.
In one aspect, the present invention provides wearable compression
devices for promoting regeneration of a tissue in a subject. The
devices comprise a surrounding member configured to encircle a body
part including a site of tissue damage and apply cyclic mechanical
compressions to the tissue at the site of tissue damage; and a
controller configured to generate the cyclic mechanical
compressions in the surrounding member.
In some embodiments, the controller comprises a microcontroller
configured to control one or more of the following: a frequency of
compression cycles, a total duration of compression cycles, a
length of a period of increasing compression in a single cycle, a
length of a period of decreasing compression in a single cycle, or
a peak compression level.
In some embodiments, the surrounding member includes one or more
inflatable portions. In other embodiments, the one or more
inflatable portions include one or more of a balloon, a bladder or
an independently inflatable member.
In some embodiments, the controller further comprises a pump
configured to provide fluid in the form of a liquid or a gas to at
least some of the one or more inflatable portions of the
surrounding member.
In some embodiments, the controller further comprises a valve
configured to control fluid flow between the pump and at least some
of the one or more inflatable portions of the surrounding member,
wherein the valve is controlled by the microcontroller.
In some embodiments, the devices further comprise a pressure sensor
disposed in or on the surrounding member configured to be
positioned between at least one of the one or more inflatable
portions of the surrounding member and the site of tissue damage,
the pressure sensor in communication with the microcontroller.
In some embodiments, the controller includes storage configured to
store information from a pressure sensor regarding a plurality of
pressure measurements during application of cyclic mechanical
compressions to tissue of a subject.
In other embodiments, the controller includes storage storing
machine readable instructions for: applying an inflation pressure
to at least some of the one or more inflatable portions of the
surrounding member to generate the cyclic mechanical compressions
in the surrounding member; receiving information regarding a
pressure measurement from the pressure sensor; and modifying one or
both of a level of the inflation pressure applied or a time period
that the inflation pressure is applied to at least some of the one
or more inflatable portions based on the information regarding a
pressure measurement received from the pressure sensor.
In some embodiments, the controller includes storage storing
machine readable instructions for cyclically applying an inflation
pressure to at least some of the one or more inflatable portions of
the surrounding member to generate the cyclic mechanical
compressions in the surrounding member.
In some embodiments, the surrounding member comprises one or more
soft actuators. In some embodiments, the one or more soft actuators
include one or more of a fiber reinforced actuator, a Pneunet
bending actuator, a McKibben actuator, or a pleated air muscle.
In some embodiments, the surrounding member further comprises an
inflatable portion having a channel, and wherein at least one of
the one or more soft actuators is configured to be disposed in the
channel.
In some embodiments, the compression device is configured to apply
the cyclic mechanical compressions to the tissue while being worn
and is self-contained.
The present invention is illustrated by the following drawings and
detailed description, which do not limit the scope of the invention
described in the claims.
BRIEF DESCRIPTION THE DRAWINGS
FIGS. 1A-C depict the cyclic mechanical compressions generated by
biphasic ferrogels and pressure cuffs. Specifically, FIG. 1A
depicts the experimental design showing injury, implant, and
stimulation profile. FIG. 1B depicts the schematic of biphasic
ferrogel implant in mouse hindlimb showing orientation of ferrogel
relative to skin, muscle tissue, and magnet (left). Pressure
profile of biphasic ferrogel undergoing repeated magnetic
stimulations (right). FIG. 1C depicts the schematic of pressure
cuff on mouse hindlimb showing orientation of balloon and
polycarbonate cuff relative to skin and muscle tissue (left).
Pressure profile of balloon cuff undergoing repeated inflations and
deflations (right).
FIGS. 2A and 2B depict that magnetic stimulation of ferrogel
implants decreases fibrous capsule thickness. Specifically, FIG. 2A
depicts the cross-sections of biphasic ferrogels stained with
hematoxylin and eosin at 3 days and 2 weeks following implantation.
Skin (X), fibrous capsule (*), and ferrogels (F) are indicated. It
is important to note that significant fibrous capsule formation was
not observed at 3 days in either ferrogel condition and surrounding
tissues were often lost during processing. FIG. 2B depicts
quantified fibrous capsule thickness of non-stimulated and
stimulated biphasic ferrogels following 2 weeks of implantation.
Fibrous capsule boundaries are marked with red dashed lines in FIG.
2A. Scale bar represents 500 .mu.m. Data were compared using a
two-tailed unpaired Student's t-test with Welch's correction (N=9,
*p<0.05). Error bars represent standard deviations.
FIGS. 3A-C depict that ferrogel stimulation leads to improved
muscle regeneration. Specifically, FIG. 3A depicts histological
cross-sections of tibialis anterior muscles stained with
hematoxylin and eosin 3 days and 2 weeks following no treatment (No
Treat), treatment with a pressure cuff (Press Cuff), treatment with
a non-stimulated biphasic ferrogel (Gel No Stim), or treatment with
a stimulated biphasic ferrogel (Gel Stim). Scale bar represents 100
m. FIG. 3B depicts quantification of myofibers residing in the
defect containing centrally located nuclei 2 weeks post-treatment.
Values are expressed as a percentage of the total number of
myofibers in the defect. FIG. 3C depicts quantified mean muscle
fiber size in the defect area 3 days and 2 weeks post-treatment.
Data were compared using ANOVA with Bonferroni's post-hoc test
(N=5, *p<0.05). Error bars represent standard deviations.
FIGS. 4A-F depict that ferrogel stimulation decreases inflammation
and fibrosis. Specifically, FIGS. 4A and 4D depict representative
images and quantification of the inflammatory infiltrate (INFLAM)
in histological cross-sections of tibialis anterior muscles stained
with hematoxylin and eosin 2 weeks post-treatment. FIGS. 4B and 4E
depict representative images and quantification of tissue collagen
(COLL) from picosirius red stained cross-sections 2 weeks
post-treatment. FIGS. 4C and 4F depict representative images and
quantification of M1 macrophages from CCR7 stained cross-sections 2
weeks post-treatment. All values are expressed as a percentage of
the total cross-section area (A.sub.cross) of the tissue section.
All scale bars represent 200 .mu.m. Data were compared using ANOVA
with Bonferroni's post-hoc test (N=10, *p<0.05) in FIG. 4D and
Dunnet's post-hoc test (N=5, *p<0.05) in FIGS. 4E and 4F. Error
bars represent standard deviations.
FIGS. 5A-D depict that intramuscular oxygen concentration increases
during ferrogel and pressure cuff stimulation. Specifically, FIG.
5A depicts quantified perfusion of injured hindlimbs normalized to
contralateral controls, as measured by Laser doppler perfusion
imaging (LDPI). A difference between the stimulated and
non-stimulated biphasic ferrogel conditions appeared at day 9 is
indicated with a (*). FIG. 5B depicts quantified capillary density
in injured muscle, as assessed by CD31+ staining 2 weeks
post-treatment. FIG. 5C depicts representative oxygen probe trace
with stimulation period marked by a dashed line. FIG. 5D depicts
the LPCI images of perfusion of injured hindlimbs before (left) and
after (right) treatment of pressure cuff. Data were compared using
ANOVA with Bonferroni's post-hoc test (N=5, *p<0.05). Error bars
represent standard deviations.
FIG. 6 depicts that cyclic mechanical compressions enhance
functional muscle regeneration. Maximum contractile force following
tetanic stimulation of injured muscles 2 weeks post-treatment.
Force measurements were normalized to muscle wet weight. Data were
compared using ANOVA with Dunnet's post-hoc test (N=5-10,
*p<0.05, ***p<0.001).
FIGS. 7A and 7B depict that the biphasic ferrogels exhibit
fatigue-resistance. Percent decrease in biphasic ferrogel (FIG. 7A)
Young's modulus and (FIG. 7B) toughness at 50% strain following
8400 cyclic compressions to 50% strain, as compared with the values
calculated from the first cycle of compression. Values represent
the mean and standard deviation (N=4).
FIG. 8 depicts a schematic for a compression device including a
pump, a valve, a microcontroller, a sleeve and a balloon.
FIG. 9 depicts recovery of muscle tetanic force in response to
pressure cuff-mediated mechanical stimulation. The average
contractile force of injured tibialis anterior muscle was measured
24 hours after ischemia surgery (Day 1) without any treatment or
after 3, 7, and 14 days with and without mechanical stimulation.
Control (Ctrl) indicates non-treated control group. Data were
compared using ANOVA with Bonferroni's post-hoc test (N=5-9,
*p<0.05).
FIG. 10 depicts changes in cytokine levels from the injured muscle
by mechanical stimulation. Levels of listed cytokines were screened
on the injured muscle treated with and without mechanical
stimulation for 7 days after ischemic injury. The intensity of
color indicates the degree of difference.
FIG. 11 depicts kinetics of fluorescent intensities from
intramuscularly injected dextran in response to mechanical
stimulation. Changes in signals of fluorescently labeled dextran
injected into tibialis anterior muscle were measured post-injection
and post-mechanical stimulation (from 5 to 60 minutes). Control
(ctrl) indicates non-treated control group. Data were compared
using Student's t-test, (N=3, *p<0.05).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based, at least in part, on the discovery
that devices with a capacity to apply cyclic compression forces
such as biphasic ferrogels and pressure cuffs can be used to
mechanically stimulate and regenerate injured tissue, e.g., muscle
tissue, without the use of growth factors or cells. In particular,
both magnetic actuation of biphasic ferrogel scaffolds implanted at
the site of muscle injury and external actuation of compression
device surrounding the site of muscle injury resulted in uniform
cyclic compressions that led to reduced fibrous capsule formation
around the implant, as well as reduced fibrosis and inflammation in
the injured muscle. Furthermore, ferrogel-driven and pressure
cuff-driven mechanical compressions led to enhanced muscle
regeneration and an approximately 3- and 2.2-fold increase in
maximum contractile force of the treated muscle, respectively.
Accordingly, the present invention provides methods and
compositions for promoting regeneration of a tissue in a subject in
need thereof. Other embodiments of the invention include methods
relating to preventing or reducing inflammation of a tissue,
preventing or reducing fibrosis of a tissue, increasing a mass of a
tissue, enhancing a function of a tissue, increasing a level of
oxygen available to a tissue, increasing a rate of metabolic waste
product removal from a tissue and increasing blood perfusion of a
tissue in a subject in need thereof. In a further embodiment, the
invention includes methods for treating a severe muscle tissue
damage in a subject in need thereof.
I. Definitions
In order that the present invention may be more readily understood,
certain terms are first defined. In addition, it should be noted
that whenever a value or range of values of a parameter are
recited, it is intended that values and ranges intermediate to the
recited values are also intended to be part of this invention.
In the following description, for purposes of explanation, specific
numbers, materials and configurations are set forth in order to
provide a thorough understanding of the invention. It will be
apparent, however, to one having ordinary skill in the art that the
invention may be practiced without these specific details. In some
instances, well-known features may be omitted or simplified so as
not to obscure the present invention. Furthermore, reference in the
specification to phrases such as "one embodiment" or "an
embodiment" means that a particular feature, structure or
characteristic described in connection with the embodiment is
included in at least one embodiment of the invention. The
appearances of phrases such as "in one embodiment" in various
places in the specification are not necessarily all referring to
the same embodiment.
The articles "a" and "an" are used herein to refer to one or to
more than one (i.e., to at least one) of the grammatical object of
the article. By way of example, "an element" means one element or
more than one element.
As used herein the term "comprising" or "comprises" is used in
reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
The term "consisting of" refers to compositions, methods, and
respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
Other than in the operating examples, or where otherwise indicated,
all numbers expressing quantities of ingredients or reaction
conditions used herein should be understood as modified in all
instances by the term "about." The term "about" when used in
connection with percentages may mean.+-.1%. Furthermore, the term
"about" can mean within .+-.1% of a value.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of this
disclosure, suitable methods and materials are described below. The
term "comprises" means "includes." The abbreviation, "e.g." is
derived from the Latin exempli gratia, and is used herein to
indicate a non-limiting example. Thus, the abbreviation "e.g." is
synonymous with the term "for example."
The term "cyclic mechanical compression" refers to the repeated
cyclic application of pressure followed by a release of that
pressure that, in turn, results in a compression and a
decompression of the target tissue.
By "treatment", "prevention" or "amelioration" of a disease or
disorder is meant delaying or preventing the onset of such a
disease or disorder, reversing, alleviating, ameliorating,
inhibiting, slowing down or stopping the progression, aggravation
or deterioration, the progression or severity of a condition
associated with such a disease or disorder. In one embodiment, the
symptoms of a disease or disorder are alleviated by at least 5%, at
least 10%, at least 20%, at least 30%, at least 40%, or at least
50%.
As used herein, the term "polymer" is intended to include both
oligomeric and polymeric species, i.e., compounds which include two
or more monomeric units, which may be a homopolymer or a copolymer.
The term "homopolymer" is a polymer incorporating a single species
of monomer units. The term "copolymer" is a polymer constructed
from two or more chemically distinct species of monomer units in
the same polymer chain. A "block copolymer" is a polymer which
incorporates two or more segments of two or more distinct species
of homopolymers or copolymers.
As used herein, the term "swelling agent" refers to those compounds
or substances which affect at least a degree of swelling.
Typically, swelling agents is an aqueous solution or organic
solvent, however swelling agent can also be a gas. In some
embodiments, swelling agent is water or a physiological solution,
e.g. phosphate buffer saline, or growth media.
As used herein, a "subject" means a human or animal. Usually the
animal is a vertebrate such as a primate, rodent, domestic animal
or game animal. Primates include chimpanzees, cynomologous monkeys,
spider monkeys, and macaques, e.g., Rhesus. Rodents include mice,
rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game
animals include cows, horses, pigs, deer, bison, buffalo, feline
species, e.g., domestic cat, canine species, e.g., dog, fox, wolf,
avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout,
catfish and salmon. Patient or subject includes any subset of the
foregoing, e.g., all of the above, but excluding one or more groups
or species such as humans, primates or rodents. In certain
embodiments, the subject is a mammal, e.g., a primate, e.g., a
human. The terms, "patient" and "subject" are used interchangeably
herein. Preferably, the subject is a mammal. The mammal can be a
human, non-human primate, mouse, rat, dog, cat, horse, or cow, but
this term is not limited to these examples. Mammals other than
humans can be advantageously used as subjects that represent animal
models of muscle injury or damage, or other related pathologies. In
addition, the methods described herein can be used to treat
domesticated animals and/or pets. A subject can be male or female.
A subject can be one who has been previously diagnosed with or
identified as suffering from or having tissue injury or damage,
e.g., muscle tissue injury or damage, or having one or more
complications related to such tissue injury or damage. Optionally,
such subjects have not already undergone treatment for the tissue
injury damage.
As used herein the term "surrounding a site of tissue damage"
refers to, at least, encircling the site of tissue damage, but does
not require that the site of tissue damage be surrounded on all
sides. For example, it does not require that the tissue damage be
surrounded over all of the polar angle of inclination from 0 to 180
degrees and all of the azimuthal angle of 0 to 360 degrees
extending outward from the center of the site of tissue damage. In
one embodiment, the term includes surrounding the tissue damage
over all sides, for example, over all of the polar angle of
inclination from 0 to 180 degrees and all of the azimuthal angle of
0 to 360 degrees extending outward from the center of the site of
tissue damage.
II. Methods of the Invention
In one aspect, the invention provides methods for promoting
regeneration of a tissue, e.g., muscle tissue, in a subject in need
thereof. The methods of the present invention include contacting
the tissue, e.g., muscle tissue, with a composition suitable for
applying cyclic mechanical compressions, and applying cyclic
mechanical compression to the tissue, e.g., muscle tissue, thereby
promoting regeneration of the tissue, e.g., muscle tissue.
As used herein, the term "contacting" (e.g., contacting a tissue,
e.g., a muscle tissue, or a plurality of tissues with a
composition) is intended to include any form of interaction (e.g.,
direct or indirect interaction) of a composition and a tissue or a
plurality of tissues. The term contacting includes incubating a
composition and a tissue or a plurality of tissues together, e.g.,
injecting or implanting the composition to a tissue or a plurality
of tissues in a subject or placing the composition on a tissue or a
plurality of tissues together in a subject, e.g., by configuring
the composition to be disposed externally to the body and
surrounding a tissue or a plurality of tissues from at least a
portion of a body part (e.g., a limb, a spine, a neck, a waist, a
shoulder, a knee, a joint, an ankle, a calf, a thigh, a foot, a
hand, a wrist, an arm, a should or an axilla), or by configuring
the composition to be disposed, at least partially, internally
within the body and to, at least partially, surround a tissue or a
plurality of tissues from a body part (e.g. an esophagus, a
urethral sphincter or an anal sphincter).
The term "cyclic mechanical compression" refers to the repeated
cyclic application of pressure followed by a release of that
pressure which, in turn, results in a compression and a
decompression of the target tissue, e.g., a muscle tissue, a bone
tissue, or an endothelial tissue.
In some embodiments, the target tissue is intact. In other
embodiments, the target tissue is damaged. In some embodiments, the
site of tissue damage is on or in a limb, a spine, a neck, a waist,
a shoulder, a knee, or a joint of a subject. In some embodiments,
the limb is a lower limb of the subject and the site of tissue
damage is on or in an ankle, a calf, a thigh or a foot. In other
embodiments, the limb is a upper limb of the subject and the site
of tissue damage is on or in a hand, a wrist, an arm, a shoulder or
an axilla. In some embodiments, the site of tissue damage is within
an esophagus of a subject. In other embodiments, the site of tissue
damage is within an urethral or anal sphincter of a subject.
In some embodiments, the tissue, e.g., muscle tissue, is contacted
with the composition suitable for applying cyclic mechanical
compression at the site of tissue damage after the damage has
occurred. In some embodiments, cyclic mechanical compressions are
applied to the site of tissue damage immediately after the damage
has occurred. For example, cyclic mechanical compressions are
applied to the site of tissue damage within less than 5, 10, 20,
30, 40, 50, 60 minutes after the damage has occurred. In other
embodiments, cyclic mechanical compressions are applied to the site
of tissue damage at least 1, 2, 3, 4, 5, 7, 8, 9, 10, 12 or 24
hours after the damage has occurred. In some embodiments, cyclic
mechanical compressions are applied to the site of tissue damage at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or at least 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 11, 12, 24, 48 or 60 months after the damage has
occurred. In other embodiments, cyclic mechanical compressions are
applied to the site of tissue damage at least 1, 2, 3, 4, 5, 6
years after the damage has occurred.
In some embodiments, cyclic mechanical compressions, generated by
the composition suitable for applying cyclic mechanical
compression, are applied to the tissue, e.g., a muscle tissue, at
the site of tissue damage over a period of time, for example, for
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, 60, 72,
84, 96 or 120 hours. In other embodiments, cyclic mechanical
compressions are applied to the site of tissue damage for about 1
to 30 days, about 1 to 50 days, about 1 to 100 days, about 1 to 200
days or about 1 to 300 days. In certain embodiments, the cyclic
mechanical compressions are applied to the site of tissue damage
until the tissue damage has been recovered.
In some embodiments, cyclic mechanical compressions are applied
with a peak pressure of at least 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,
700, 800, 900 or 1000 kPa. In other embodiments, cyclic mechanical
compressions are applied with a peak pressure of about 0.1 kPa to
1000 kPa, about 1-10 kPa, about 1-20 kPa, about 1-30 kPa, about
1-50 kPa, about 1-100 kPa, about 1-1000 kPa, about 10-100 kPa,
about 10-200 kPa, about 10-300 kPa, about 10-500 kPa, about 10-1000
kPa, about 100-1000 kPa, about 200-1000 kPa, about 300-1000 kPa,
about 400-1000 kPa, about 500-1000 kPa, about 600-1000 kPa, about
700-1000 kPa, about 800-1000 kPa, or about 900-1000 kPa.
In some embodiments, cyclic mechanical compressions are applied at
least once daily. In other embodiments, cyclic mechanical
compressions are applied at least three times daily.
Cyclic mechanical compressions applied by the composition suitable
for the methods of the present invention can be generated by any
known methods in the art, for example, by an electromagnetic
signal. The electromagnetic signal, which causes the composition to
apply cyclic mechanical compression to the tissue, can be produced
by an electronic signal generator. The term "electronic signal
generator", as used herein, may include an electromagnet or
electrically-polarizable element, or at least one permanent magnet.
In some embodiments, the electromagnetic signal can be produced at
least in part according to a pre-programmed pattern. The
electromagnetic signal may have a defined magnetic field strength
or spatial orientation, or a defined electric field strength or
spatial orientation. In some embodiments, the electromagnetic
signal is generated by application of a magnetic field. In other
embodiments, the electromagnetic signal has a defined magnetic
field strength.
As used herein, the term "magnetic field" refers to magnetic
influences which create a local magnetic flux that flows through
the composition and can refer to field amplitude,
squared-amplitude, or time-averaged squared-amplitude. It is to be
understood that magnetic field can be a direct-current (DC)
magnetic field or alternating-current (AC) magnetic field. Magnetic
field strength can range from about 0.001 Tesla to about 1 Tesla.
In some embodiments, magnetic field strength is in the range from
about 0.01 Tesla to about 1 Tesla. In some other embodiments,
magnetic field strength is in the range from about 0.1 Tesla to
about 1 Tesla. In other embodiments, the magnetic field strength is
about 0.5 Tesla.
Cyclic mechanical compressions applied by the composition suitable
for the methods of the present invention can also be generated by a
device, for example, a compression device comprising a surrounding
member configured to encircle a body part, and a controller
configured to generate cyclic mechanical compression in the
surrounding member.
As used herein, a "controller" is a device or system configured to
generate cyclic mechanical compressions and to control properties
of the cyclic mechanical compressions, such as, the frequency, the
amplitude and/or the duration of the cyclic mechanical compressions
being generated. In some embodiments, the controller includes a
microcontroller that controls the properties of the cyclic
mechanical compressions. In some embodiments, the controller
includes storage for holding machine readable instructions,
measurements of pressure from a pressure sensor, or other
information. In some embodiments, microcontroller includes storage
for holding machine readable instructions, measurements of pressure
from a pressure sensor, or other information. In some embodiments,
the controller includes additional storage that is not included in
the microcontroller, but is accessible to the microcontroller for
holding machine readable instructions, measurements of pressure
from a pressure sensor, or other information.
Existing treatments for tissue regeneration have mainly focused on
the delivery of biologics, e.g., relying on the use of cell and
growth factor-based approaches. The methods of the present
invention, however, are based, at least in part, on the surprising
discovery that mechanical stimulation alone, e.g., cyclic
mechanical compressions, on the target tissue is sufficient to
enhance tissue repair. For example, the development of a
composition that can apply cyclic mechanical compressions to a
damaged tissue, e.g., a muscle tissue, without the use of growth
factors or cells, can be used to mechanically stimulate and
regenerate damaged tissue, representing a novel therapeutic
strategy for treatment of tissue injuries.
The term "tissue regeneration", as used herein, encompasses both
regeneration of tissue with recourse to exactly the type of tissue
to be regenerated, in the sense of an increase in the mass of the
tissue, as well as the production of new tissue starting from a
different type of tissue or cell than that to be produced. For
example, the term "muscle regeneration" refers to the process by
which new muscle fibers form from muscle progenitor cells. The
growth of muscle may occur by the increase in the fiber size and/or
by increasing the number of fibers. The growth of muscle may be
measured by an increase in weight, an increase in protein content,
an increase in the number of muscle fibers, or an increase in
muscle fiber diameter. An increase in growth of a muscle fiber can
be defined as an increase in the diameter where the diameter is
defined as the minor axis of ellipsis of the cross section.
The tissue, e.g., muscle tissue, regenerated based on the methods
of the present invention is functional. The functional quality of
the regenerated muscle tissue can be measured by any methods known
in the art. For example, as demonstrated in Example 6 of the
present invention, the function of the regenerated muscle tissue
can be measured by the contractile force of each regenerated
muscle. Peak tetanic force can be determined as the difference
between the maximum force during contraction and the baseline
level. An increase in the maximum contractile force of muscle
tissue upon treatment with cyclic mechanical compressions indicates
that the regenerated muscle tissue is functional. The increase in
the maximum force of the regenerated muscle tissue can be, for
example, at least 0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold,
3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 10-fold, 20-fold,
30-fold or 50-fold.
The methods of the present invention are suitable for promoting
regeneration of any type of tissue, such as external epithelial
tissue, internal epithelial tissue, endothelial tissue or
mesenchymal tissue. Examples of tissue that can be regenerated
based on the methods of the present invention may include, but not
limited to, heart tissue, blood vessel tissue, skin tissue, muscle
tissue, bone tissue, cartilage tissue, connective tissue, tendon
tissue, and ligament tissue.
In some embodiments, the methods of the present invention are
suitable for promoting regeneration of a muscle tissue. The phrase
"muscle tissue", as used herein, encompasses any mammalian muscle
tissue. In some embodiments, the muscle tissue is a human muscle
tissue. In other embodiments, the muscle tissue is a muscle tissue
from a domesticated animal, a pet or a game animal, for example, a
dog muscle tissue, a horse muscle tissue, a cow muscle tissue, or a
rabbit mouse tissue. In some embodiments, the muscle tissue is
selected from the group consisting of a smooth muscle tissue, a
skeletal muscle tissue and a cardiac muscle tissue. Skeletal muscle
tissue is under voluntary control. The muscle fibers are syncytial
and contain myofibrils, tandem arrays of sarcomeres. Smooth muscle
tissue is made up from long tapering cells, generally involuntary
and differs from striated muscle in the much higher actin/myosin
ratio, the absence of conspicuous sarcomeres and the ability to
contract to a much smaller fraction of its resting length. Smooth
muscle cells are found particularly in blood vessel walls,
surrounding the intestine and in the uterus. Cardiac muscle tissue
is a striated but involuntary tissue responsible for the pumping
activity of the vertebrate heart. The individual cardiac muscle
cells are not fused together into multinucleate structures as they
are in striated muscle tissue. As used herein, the phrases "cardiac
muscle tissue" and "myocardium tissue" are interchangeable.
In some embodiments, the muscle tissue is damaged. As used herein,
the terms "damaged muscle tissue" or "muscle tissue damage" refer
to a muscle tissue, such as a skeletal or cardiac muscle that has
been altered for instance by a physical injury or accident,
disease, infection, over-use, loss of blood circulation, or by
genetic or environmental factors such as cryo-damage. As used
herein, the term "cryo-damage" refers to damage to tissues, cells,
or other biological substrates as a result of exposure to cold. In
other embodiments, a damaged muscle tissue is a dystrophic muscle
or an aging muscle.
In some embodiments, the muscle tissue damage is a severe muscle
tissue damage. A severe muscle tissue damage refers to a muscle
mass loss or injury of greater than about 20%, e.g., about greater
than 25%, 30%, 35%, 40%, 45% or 50%. Severe muscle tissue injures
resulting in a muscle mass loss or injury of greater than about 20%
can lead to extensive fibrosis and loss of muscle function.
In some embodiments, muscle tissue damage is induced by mechanical
injury, such as acute and chronic strains. In other embodiments,
muscle tissue damage is induced by exercise or muscle laceration,
e.g., post-exercise muscular cramp. In some embodiments, muscle
tissue damage is induced by loss of muscle tissue due to disease
such as myopathy. Without limitation, myopathy can be a congenital
myopathy or an acquired myopathy. Exemplary myopathies include, but
are not limited to, dystrophies, myotonia (neuromytonia),
congenital myopathies (e.g., nemaline myopathy, multi/minicore
myopathy, centronuclear myopathy (or myotubular myopathy)),
inflammatory myopathies, metabolic myopathies (e.g., glycogen
storage disease and lipid storage disorder), dermatomyositis,
polymyositis inclusion body myositis, myositis ossificans,
rhabdomyolysis and myoglobinuirias.
In some embodiments, myopathy is a dystrophy selected from the
group consisting of muscular dystrophy, Duchenne muscular
dystrophy, Becker's muscular dystrophy, reflex sympathetic
dystrophy, detinal dystrophy, conal dystrophy, myotonic dystrophy,
corneal dystrophy, and any combinations thereof.
In some embodiments, muscle tissue damage is induced by muscle
hypertrophy. In other embodiments, muscle tissue damage is induced
by muscle atrophy or wasting, e.g., muscle wasting from
post-surgery bed rest. In some embodiments, muscle tissue damage is
induced by genetic disorders such as, but not limited to muscular
dystrophies. In other embodiments, muscle tissue damage is induced
by chronic disorders such as, but not limited to AIDS, cancer,
chronic heart failure, and kidney disease. In some embodiments,
muscle tissue damage is induced by diseases related to aging.
In some embodiments, muscle tissue damage is induced by diseases
related to inflammation of connective tissues surrounding muscle,
for example, fasciitis. As used herein, the term "fasciitis" refers
to an inflammation of fascia, which is the connective tissue
surrounding muscles, blood vessels and nerves. Exemplary fasciitis
diseases include, but not limited to, plantar fasciitis which is
one of the most common causes of pain in heel and the bottom of the
foot; eosinophilic fasciitis which is a disorder that results in
pain and inflammation in arms and legs, and necrotizing fasciitis
which involves infection of deeper layers of skin and subcutaneous
tissues.
In some embodiments, the muscle tissue damage is induced by toxin.
In other embodiments, the muscle tissue damage is induced by
ischemia. In some embodiments, the muscle tissue damage is induced
by a combination of toxin and ischemia. In other embodiments, the
muscle tissue damage is induced by hind limb ischemia. In some
embodiments, the muscle tissue damage is induced by trauma.
Exemplary symptoms of muscle damage include, but are not limited
to, swelling, bruising or redness, open cuts as a consequence of an
injury, pain at rest, pain when specific muscle or the joint in
relation to that muscle is used, weakness of the muscle or tendons,
and an inability to use the muscle at all.
The principle of using mechanical stimulation, e.g., cyclic
mechanical compressions, to enhance tissue regeneration can be
applied to any type of tissue. In some embodiments, the methods of
the present invention are suitable for promoting regeneration of an
endothelial tissue. Endothelial tissue or cells make up the
structure of blood vessels. Endothelial cells are involved in many
aspects of vascular biology such as angiogenesis, e.g., formation
of new blood vessels, repair of damaged or disease organs, and
control of blood pressure through vasoconstriction and
vasodilation. Accordingly, regeneration of endothelial cells is
critical for angiogenesis and organ repair.
In other embodiments, the methods of the present invention are
suitable for promoting regeneration of a bone tissue. Bone tissue
regeneration is often required in conditions such as skeletal
reconstruction of large bone defects created by trauma, infection,
tumor resection and skeletal abnormalities, or cases in which
regenerative process is comprised, including avascular necrosis and
osteoporosis.
In one aspect, the invention provides a method for increasing a
mass of a tissue in a subject in need thereof. An increase in the
mass of a tissue represents a marker for successful tissue
regeneration. The method of the present invention comprises
contacting the tissue with a composition suitable for applying
cyclic mechanical compressions, and applying cyclic mechanical
compression to the tissue, thereby increasing the mass of the
tissue.
The methods of the present invention are suitable for increasing
the mass of any type of tissue, such as external epithelial tissue,
internal epithelial tissue, endothelial tissue or mesenchymal
tissue. Examples of tissue that can be regenerated based on the
methods of the present invention may include, but not limited to,
heart tissue, blood vessel tissue, skin tissue, muscle tissue, bone
tissue, cartilage tissue, connective tissue, tendon tissue, and
ligament tissue.
In some embodiments, the methods of the present invention are
suitable for increasing the mass of a muscle tissue. The mass of a
muscle tissue can be enhanced by increasing the mitogenesis,
myogenesis, differentiation, or survival of muscle cells in a
subject, for example, a mammal, i.e., a human. The methods of the
present invention are also suitable to slow or halt net muscle loss
or to increase the amount or quality of muscle present in a
subject.
The term "muscle cell", as used herein, refers to any cell which
contributes to muscle tissue. Myoblasts, satellite cells, myotubes,
and myofibril tissues are all included in the term "muscle cells"
and may all be regenerated using the methods of the invention.
Mitogenesis, as used herein, refers to any cell division which
results in the production of new muscle cells in the subject.
Mitogenesis may be induced in muscle cells of skeletal muscle,
smooth muscle or cardiac muscle. Mitogenesis in vitro can be
measured by any methods known in the art. For example, mitogenesis
can be assayed by exposing cells to a labeling agent for a time
equivalent to two doubling times, and calculating the mitotic
index. The mitotic index is the fraction of cells in the culture
which have labeled nuclei when grown in the presence of a tracer,
i.e., BrdU, which only is incorporated during S phase of the cell
cycle, and the doubling time is defined as the average time
required for the number of cells in the culture to increase by a
factor of two. Mitogenesis is defined by an increase in mitotic
index relative to untreated cells of at least 50%, e.g., 60%, 70%,
80%, 90%, 100%, 150%, 200%, 250% or 300%.
Myogenesis, as used herein, refers to any fusion of myoblasts to
yield myotubes. An effect on myogenesis is defined as an increase
in the fusion of myoblasts and the enablement of the muscle
differentiation program, which is characterized as a fusion index
in vitro. The fusion index is defined as the fraction of nuclei
present in multinucleated cells in the culture relative to the
total number of nuclei present in the culture. Myogenesis may also
be determined by assaying the number of centrally located nuclei
per area in myotubes, as demonstrated in Example 3 of the present
invention, or by measurement of the levels of muscle specific
protein by Western analysis.
The survival of muscle fibers, as used herein, refers to the
prevention of loss of muscle fibers as evidenced by necrosis or
apoptosis or the prevention of other mechanisms of muscle fiber
loss. Survival as used herein indicates an decrease in the rate of
cell death of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100% relative to an untreated control. The rate of survival may be
measured by counting cells stainable with a dye specific for dead
cells (such as propidium iodide) in culture when the cells are a
couple days post-differentiation.
In another aspect, the invention provides methods for increasing a
function of a tissue in a subject in need thereof. These methods
include contacting the tissue with a composition suitable for
applying cyclic mechanical compressions, and applying cyclic
mechanical compression to the tissue, thereby increasing the
function of the tissue in the subject.
The functional quality of the regenerated tissue, e.g., a muscle
tissue, can be measured by any methods known in the art. For
example, as demonstrated in Example 6 of the present invention, the
function of the regenerated muscle tissue can be measured by the
contractile force of each regenerated muscle. Peak tetanic force
can be determined as the difference between the maximum force
during contraction and the baseline level. An increase in the
maximum contractile force of muscle tissue upon treatment with
cyclic mechanical compressions indicates that the regenerated
muscle tissue is functional. The increase in the maximum force of
the regenerated muscle tissue can be, for example, at least
0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold,
4-fold, 4.5-fold, 5-fold, 10-fold, 20-fold, 30-fold or 50-fold.
In yet another aspect, the invention provides methods for
preventing or reducing inflammation of a tissue in a subject in
need thereof. These methods include contacting the tissue with a
composition suitable for applying cyclic mechanical compressions,
and applying cyclic mechanical compression to the tissue, thereby
preventing or reducing inflammation of the tissue.
The term "inflammation" refers to a part of the complex biological
response of body tissues to harmful stimuli, such as pathogens,
damaged cells, irritants, ischemic and toxic assaults. The purpose
of inflammation is to eliminate the initial cause of cell injury,
clear out necrotic cells and tissues damaged from the original
insult and the inflammatory process, and to initiate tissue repair.
Inflammation can be classified as either acute or chronic. Acute
inflammation is the initial response of the body to harmful stimuli
and is achieved by the increased movement of plasma and leukocytes
from the blood into the injured tissues. Chronic inflammation leads
to a progressive shift in the type of cells present at the site of
inflammation and is characterized by simultaneous destruction and
healing of the tissue from the inflammatory process. As a result,
the inflammatory response represents a two-sided sword: beneficial
in terms of the repair process to injury; detrimental when
proceeding in an uncontrolled manner, which then leads to
progressive fibrosis with a loss of function. Thus, controlling
excessive inflammation would be of great potential therapeutic
benefit for inhibiting progressive fibrosis during tissue
injury.
Fibrosis, as used herein, refers to a process of wound healing and
repair that is activated in response to injury and is associated
with chronic inflammatory diseases. A key event leading to fibrous
capsule formation is the adhesion of immune cells, such as
macrophages, to the site of injury, and these cells secrete
proteins that modulate fibrosis leading to the proliferation and
activation of fibroblasts, which results in an excessive deposition
of extracellular matrix in the surrounding tissue and impairs the
architecture and function of the underlying tissue.
Inflammation can be characterized by signs or manifestations
comprising redness, swelling, heat, pain, and the loss of function
of the involved tissue. The presence of inflammation in the
involved tissue indicates the presence of injury or disease, while
the amount of inflammation in the injured, damaged or deformed
tissue is directly proportional to the amount of damage or disease
in that tissue and is inversely proportional to the degree of
healing in the same tissue. Various methods and equipment have been
developed for assessing the presence or absence of inflammation in
tissues. Such methods and equipment include performing a physical
examination of the involved tissue; blood tests, such as
erythrocyte sedimentation rate or C-reactive protein level;
radiographic tests, such as plain X-rays or magnetic resonance
imaging (MRI); and research procedures, such as thermography.
Additional methods for monitoring the presence or absence of
inflammation in tissues include examining the level of tissue
scarring and fibrosis using histologic sections. For example, the
extent of tissue fibrosis can be assessed by visualizing picosirius
red stained collagen I and III under polarized light.
Alternatively, the extent of tissue fibrosis can be determined by
quantification of inflammatory infiltrate at the site of
injury.
Compositions suitable for use in the methods of the present
invention are suitable for injection, implantation or disposing
internally within the body, e.g., at the site of tissue damage. In
some embodiments, the compositions are configured to surround or
encircle a body part or at least a portion of a body part (e.g., an
esophagus, a urethral sphincter, or an anal sphincter). In other
embodiments, the compositions suitable for use in the methods of
the present invention are implanted or disposed internally within
the body, at a site of tissue damage upon major surgery to assist
tissue regeneration or to treat tissue injury.
Compositions suitable for use in the methods of the present
invention are also suitable to be disposed externally to the body
and surround at least a portion of a body part (e.g., a limb, a
spine, a neck, a waist, a shoulder, a knee, a joint, an ankle, a
calf, a thigh, a foot, a hand, a wrist, an arm, a shoulder, or an
axilla).
Current clinical devices are often plagued by the formation of
thickened tissue capsules upon implantation at the site of
interaction between the mechanical components and the body tissues.
The ability to inhibit fibrous capsule formation with cyclic
mechanical compressions has a great potential utility for
implantable drug delivery devices and sensors that require
unobstructed diffusion around the implant for proper function. As
demonstrated in Example 4 of the present invention, application of
cyclic mechanical compressions at the injured tissue resulted in a
reduction in fibrous capsule thickness and a decrease amount of
inflammatory cells at the site of implant, thus alleviating the
overall inflammation in the target tissue.
Accordingly, in one aspect, the invention provides methods for
preventing or reducing fibrosis of a tissue in a subject, e.g., a
tissue at a site of implantation in a subject. The methods of the
present invention include contacting the tissue with a composition
suitable for applying cyclic mechanical compressions, and applying
cyclic mechanical compression to the tissue, thereby preventing or
reducing fibrosis of a tissue in the subject.
The principle of using mechanical stimulation, e.g., cyclic
mechanical compressions, to enhance tissue regeneration or to
reduce formation of scarring or fibrosis can be applied to any type
of tissue, such as external epithelial tissue, internal epithelial
tissue, endothelial tissue or mesenchymal tissue. Examples of
suitable tissue for this method include, but not limited to, heart
tissue, blood vessels, skin tissue, muscle tissue, bone tissue,
cartilage tissue, connective tissue, tendon tissue, and ligament
tissue.
In some embodiments, formation of fibrous capsule at the site of
tissue damage is reduced. In other embodiments, thickness of
fibrous capsule at the site of tissue damage is reduced. In yet
another embodiment, inflammatory cell removal at the site of tissue
damage is accelerated.
The direct application of cyclic mechanical compressions to the
target tissue may provide an additional convection-based benefit.
For example, enhanced fluid transportation around the site of
implant may increase the blood perfusion as well as the transport
of oxygen, nutrients, fluids to the site of injury. In addition,
the direct stimulation of target tissue may accelerate immune cell
and/or metabolic waste removal from the site of the injury, which
are all vital components of tissue health and repair. Invading
inflammatory cells near the site of injury may be expelled upon
stimulation, due to fluid convection resulting from the cyclic
mechanical compressions, leading to an overall diminished cell
presence within the site of injury.
Thus, in one aspect, the invention provides methods for increasing
a level of oxygen available to a tissue in a subject in need
thereof. These method include contacting the tissue with a
composition suitable for applying cyclic mechanical compressions,
and applying cyclic mechanical compression to the tissue, thereby
increasing the level of oxygen available to the tissue.
The oxygen level generated by the issue may be increased by
increasing blood flow to the tissue. Alternatively, the oxygen
level is increased by enhanced intramuscular convection driven by
cyclic mechanical compressions of the tissue.
In another aspect, the invention provides methods for increasing
the rate of metabolic waste product removal from a tissue in a
subject in need thereof. These method include contacting the tissue
with a composition suitable for applying cyclic mechanical
compressions, and applying cyclic mechanical compressions to the
tissue, thereby increasing the rate of metabolic waste product
removal from the tissue.
In some embodiments, the rate of metabolic waste product removal is
increased by enhanced fluid transportation driven by cyclic
mechanical compressions around the site of implantation within the
tissue.
In yet another aspect, the invention provides methods of increasing
blood perfusion to a tissue in a subject in need thereof. These
methods include contacting the tissue with a composition suitable
for applying cyclic mechanical compressions, and applying cyclic
mechanical compression to the tissue, thereby increasing blood
perfusion to the tissue.
In one aspect, the invention provides methods for promoting
regeneration of a muscle tissue in a subject suffering from muscle
damage induced by a myotoxin and ischemia. These method include
contacting the muscle tissue with a composition suitable for
applying cyclic mechanical compressions, and applying cyclic
mechanical compression to the muscle tissue, thereby promoting
regeneration of the muscle tissue.
In another aspect, the invention provides methods of treating a
severe muscle tissue damage in a subject in need thereof. These
methods include contacting the muscle tissue with a composition
suitable for applying cyclic mechanical compressions, and applying
cyclic mechanical compressions to the muscle tissue, thereby
treating the severe muscle tissue damage in the subject.
The term "treating" used herein encompasses the complete range of
therapeutically positive effects of contacting a composition
suitable for applying cyclic mechanical compressions to a tissue,
e.g., a muscle tissue, including improving the tissue function,
providing mechanical support and promoting tissue healing and
repair processes. The term treating further includes reduction of,
alleviation of, and relief of symptoms of diseases or disorders
associated with an injured or damaged tissue, e.g., a damaged
muscle tissue. In addition, the term treating includes prevention
or postponement of development of symptoms of diseases or disorders
associated with an injured or damaged tissue, e.g., a damaged
muscle tissue.
The methods of the present invention are applicable in a variety of
conditions. Exemplary diseases or disorders associated with an
injured or damaged tissue, e.g., a damaged muscle tissue, may
include, but not limited to, muscular dystrophy, Duchenne muscular
dystrophy, Becker's muscular dystrophy, reflex sympathetic
dystrophy, detinal dystrophy, conal dystrophy, myotonic dystrophy,
corneal dystrophy, fasciitis, plantar fasciitis, eosinophilic
fasciitis, necrotizing fasciitis, post-exercise muscular cramp,
pain or inflammation from cervical neck, mechanical injury such as
acute and chronic strains, myopathy, dystrophies, myotonia
(neuromytonia), congenital myopathies (e.g., nemaline myopathy,
multi/minicore myopathy, centronuclear myopathy (or myotubular
myopathy)), inflammatory myopathies, metabolic myopathies (e.g.,
glycogen storage disease and lipid storage disorder),
dermatomyositis, polymyositis inclusion body myositis, myositis
ossificans, rhabdomyolysis and myoglobinuirias, muscle hypertrophy,
muscle atrophy or wasting, e.g., muscle wasting from post-surgery
bed rest, muscle fatigue, or any diseases or disorders resulting
from a physical injury or accident, trauma, infection,
inflammation, loss of blood circulation, or by genetic or
environmental factors.
III. Compositions Suitable for Use in the Methods of the
Invention
Compositions suitable for use in the methods of the present
invention are capable of applying cyclic mechanical compressions to
a site of tissue damage, e.g., muscle tissue damage. Cyclic
mechanical compressions applied by the compositions suitable for
use in the methods of the present invention can be generated by any
known methods in the art. In some embodiment, the cyclic mechanical
compressions applied by the compositions are generated by an
electromagnetic signal. In other embodiments, the cyclic mechanical
compressions are generated by pneumatic actuation or hydraulic
actuation. In yet another embodiment, the cyclic mechanical
compressions applied by the compositions are generated by a
controller.
A. Magnetic Material-Based Compression Compositions
A magnetic material-based compression composition suitable for use
in the methods of the present invention comprises a matrix material
and a magnetic material distributed therethrough, wherein the
composition comprises pores having a mean pore diameter in range of
about 10 .mu.m to about 10000 .mu.m, wherein the magnetic material
is in the form of magnetic particles having a size in the range
from about 1 nm to about 500 nm, and wherein porosity, pore size,
pore connectivity, swelling agent concentration and/or specific
volume of the composition undergoes a change from a first value to
a second value, e.g., at least 10%, in response to an
electromagnetic signal. It is to be understood that, change in one
of porosity, pore size, pore connectivity, swelling agent
concentration, and/or specific volume can be independent of change
in one of the others, i.e., change in one may or may not effect a
change in others.
The magnetic material-based compression compositions suitable for
use in the methods of the present invention described herein can
comprise any amount of matrix and magnetic materials. For example,
the compositions can comprise 0.1-50% of the matrix material and
10-99% of the magnetic material. In some embodiments, the
compositions comprise 1-25% of the matrix material and 75-99% of
the magnetic material. Preferably, the compositions comprise 1-20%
of the matrix material and 80-90% of the magnetic material. In some
embodiments, the compositions comprise 1-15% of the matrix material
and 85-99% of the magnetic material. In other embodiments, the
compositions comprise 5-10% of the matrix material and 90-95% of
the magnetic material. The percent values can be based on weight,
volume and/or moles. In addition to the matrix and the magnetic
materials, the composition suitable for use in the methods of the
present invention can comprise additional substances such as a
swelling agent. When an additional substance is present in the
composition, matrix and magnetic material percent values are
calculated based on the matrix and magnetic material only.
Alternatively, compositions suitable for use in the methods of the
present invention can comprise 0.1%-50% of matrix material, 1-90%
of magnetic material and a swelling agent in an amount
corresponding to the balance up to 100%. Again percent values can
be based on weight, volume and/or moles. In other embodiments,
percent values are based on weight. In some embodiments, the
composition comprises 0.1%-25% of matrix material, 5-50% of
magnetic material and a swelling agent in an amount corresponding
to the balance up to 100%. In some other embodiments, the
composition comprises 0.1%-25% of matrix material, 10-50% of
magnetic material and a swelling agent in an amount corresponding
to the balance up to 100%. In some embodiments, the composition
comprises 0.1%-10% of matrix material, 10-50% of magnetic material
and a swelling agent in an amount corresponding to the balance up
to 100%. In one embodiment, the composition comprises about 1%
matrix, about 13% magnetic material and a swelling agent in an
amount corresponding to the balance up to 100%.
Ratio of matrix to magnetic material in the composition can range
from about 1:20 (matrix:magnetic) to about 20:1. The ratio can be
in the range of from about 1:120 to about 10:1, from about 1:30 to
about 5:1, or from about 1:20 to about 1:1. In some embodiments,
the ratio is in the range of about 1:20 to about 1:5. In some
embodiments, the ratio is around 1:13. In one preferred embodiment,
the ratio is in the range from about 1:1 to about 3:1. It is to be
understood that ratio can be based on weight, volume and/or
moles.
In some embodiments, the composition suitable for use in the
methods of the present invention is macroporous. As used herein,
the term "macroporous" refers to the fact that composition
comprises macropores. The composition suitable for use in the
methods of the present invention can also comprise micropores.
Generally, micropores are pores having a diameter on the order of
about 50 Angstroms or less, while macropores are pores having a
diameter on the order of about 100 Angstroms or greater. Generally,
diameter of a pore is such as to allow free flow of swelling agent
and/or a solvent through the pores. Pore diameters of compositions
described herein can range from about 20 .mu.m to 10000 .mu.m. In
some embodiments, the composition suitable for use in the methods
of the present invention comprises pores having mean pore diameter
in range from about 150 .mu.m to 1000 .mu.m, 200 .mu.m to 1000
.mu.m, 300 .mu.m to 1000 .mu.m, 400 .mu.m to 1000 .mu.m, 500 .mu.m
to 1000 .mu.m, 600 .mu.m to 1000 .mu.m, 1000 .mu.m to 10000 .mu.m,
2000 .mu.m to 10000 .mu.m, 3000 .mu.m to 10000 .mu.m, 4000 .mu.m to
10000 .mu.m, 5000 .mu.m to 10000 .mu.m, or 6000 .mu.m to 10000
.mu.m. In some embodiments, the composition suitable for use in the
methods of the present invention comprises pores having mean pore
diameter in range from about 150 .mu.m to 750 .mu.m. In some
embodiments, the composition comprises pores having mean pore
diameter of 200-750 .mu.m. In another embodiment, the composition
comprises pores having mean pore diameter of 250-700 .mu.m. In some
embodiments, the composition comprises pores having mean pore
diameter of 500-700 .mu.m. In other embodiments, the composition
comprises pores having mean pore diameter of 1000-7000 .mu.m. In
some embodiments, the composition comprises pores having mean pore
diameter of 2000-7000 .mu.m. In other embodiments, the composition
comprises pores having mean pore diameter of 3000-7000 .mu.m. In
some embodiments, the composition comprises pores having mean pore
diameter of 5000-7000 .mu.m. In other embodiments, the composition
comprises pores having mean pore diameter of about 100 .mu.m, 200
.mu.m, 300 .mu.m, 400 .mu.m, 500 .mu.m, 600 .mu.m, 700 .mu.m, 800
.mu.m, 900 .mu.m, 1000 .mu.m, 2000 .mu.m, 3000 .mu.m, 4000 .mu.m,
5000 .mu.m, 6000 .mu.m, 7000 .mu.m, 8000 .mu.m, 9000 .mu.m or 10000
.mu.m.
The changes in porosity, pore size, pore connectivity, swelling
agent concentration, and/or specific volume are preferably
reversible (i.e., porosity, pore size, pore connectivity, swelling
agent concentration, and/or specific volume detectably increases or
decreases upon application of the stimuli, and then reverts to its
original value, e.g., within 10%, 5%, 2%, 1% or less of the
original value, when the stimuli is discontinued). However, it will
be recognized that in some applications, reversibility of one or
more of porosity, pore size, pore connectivity, swelling agent
concentration, and/or specific volume is not essential. The change
in specific volume is also referred to as a volume phase transition
herein.
It is to be understood that external stimuli can be applied by
providing a stimuli that is not present, by holding back a stimuli
that is already present, or by changing the amount of a stimuli
that is already present.
In some embodiments, in response to an electromagnetic signal,
porosity, pore size, pore connectivity, swelling agent
concentration, and/or specific volume changes by at least 10%, 15%,
20%, 25% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%
or more relative to the original value. In some embodiments, in
response to an electromagnetic signal, porosity, pore size, pore
connectivity, swelling agent concentration, and/or specific volume
changes by at least 70% or more relative to the original value. In
some embodiments, the porosity, pore size, pore connectivity,
swelling agent concentration, and/or specific volume decreases in
response to the electromagnetic signal. In other embodiments, the
porosity, pore size, pore connectivity, swelling agent
concentration, and/or specific volume increases in response to the
electromagnetic signal.
As used herein, the term "porosity" means the fractional volume
(dimension-less) of the composition that is composed of open space,
e.g., pores or other openings. See for example, Coulson J. M., et.
al., Chemical Engineering (1978), volume 2, 3rd Edition, Pergamon
Press, 1978, page 126). In some embodiments, the composition has a
porosity of 0.1 to 0.99. Generally, in the absence of an external
electromagnetic signal, porosity of the composition can range from
0.5 to 0.99. Preferably porosity is in the range of from about 0.75
to about 0.99, more preferably from about 0.8 to about 0.95.
Preferably, porosity of the porous material is at least 0.75, more
preferably at least 0.8, and most preferably at least 0.9.
Several methods can be employed to measure porosity, including,
direct methods (e.g. determining the bulk volume of the porous
sample, and then determining the volume of the skeletal material
with no pores (pore volume=total volume-material volume), optical
methods (e.g., determining the area of the material versus the area
of the pores visible under the microscope, where the areal and
volumetric porosities are equal for porous media with random
structure), imbibition methods (e.g., immersion of the porous
sample, under vacuum, in a fluid that preferentially wets the
pores), water saturation method (e.g., pore volume=total volume of
water-volume of water left after soaking), water evaporation method
(e.g., pore volume in cubic centimeters=weight of saturated sample
in grams-weight of dried sample in grams), and gas expansion
methods. Methods for measuring porosity of a sample are described
in Glasbey, C. A. Horgan, G. W. and Darbyshire, J. F. J Soil Sci
(1991) 42: 479-486, contents of which are herein incorporated by
reference in their entirety.
In some embodiments, the composition suitable for use in the
methods of the present invention is a particle, e.g., a
nanoparticle or microparticle. The composition particle size will
vary depending on the particular use intended for such a particle.
In general, particles can have at least one dimension in the range
from about 1000 .mu.m to about 2000 .mu.m, 1200 .mu.m to about 2000
.mu.m, 1300 .mu.m to about 2000 .mu.m, 1500 .mu.m to about 2000
.mu.m, 1600 .mu.m to about 2000 .mu.m, 1000 .mu.m to about 1500
.mu.m, 1100 .mu.m to about 1500 .mu.m, or 1200 to about 1500
.mu.m.
Preparation of compositions suitable for use in the methods of the
present invention described herein does not require a special type
of matrix and/or magnetic materials. Any material that comprises a
spatial network structure can be used for the matrix. The matrix
can comprise materials of synthetic or natural origin (e.g.,
biopolymers) or a mixture thereof cross-linked by physical and/or
chemical interactions. In some embodiments, the matrix is not
biodegradable.
Some exemplary matrixes include swellable and non-swellable gels,
elastomers, and rubbers. Swellable gels can include hydrogels and
organogels. The term "hydrogel" indicates a cross-linked, water
insoluble, water containing material. Hydrogels have many desirable
properties for biomedical applications. For example, they can be
made nontoxic and compatible with tissue, and they are usually
highly permeable to water, ions and small molecules.
Gels generally comprise solid, cross-linked polymer networks
capable of forming a stable system in equilibrium with an
interpenetrating swelling agent. Many gel forming polymers are
known in the art. Suitable gels include polymers, copolymers, and
blockpolymers based on monomers containing ionizable groups or
polymerizable double bonds. Exemplary monomers include, but are not
limited to, acrylic acid, methyl methacrylate, methyl acrylic acid,
ethyl acrylate, vinyl sulfonic acid, styrene, styrene sulfonic acid
(e.g., p-styrene sulfonic acid), maleic acid, butenoic acid, vinyl
phosphate, vinyl phosphonate, ethylene, propylene, styrene, vinyl
methyl ether, vinyl acetate, vinyl alcohol, acrylonitrile,
acrylamide, N--(C1-C6 alkyl) acrylamide (such as
N-isopropylacrylamide, N-t-butylacrylamide), and the like. Gels are
made by homopolymerizing or copolymerizing any of the foregoing
monomers. Other suitable gel materials can include, alginate,
chitosan, collagen, gelatin, hyaluronate, fibrin, agarose, and
derivatives thereof. The gel can be a copolymer as described above
into which has been incorporated as one comonomeric component a
ligand that connects to, complexes or physically entraps the
desired magnetic material.
The gel can be cross-linked to let it take a physically stable form
when hydrated or dehydrated. Suitable cross-linking can be provided
by incorporating about 0.5 wt. % to about 1.5% wt. % of a
cross-linking agent into the gel. Cross-linking can also be
provided by incorporating about 0.01 mol % to about 15 mol % of the
cross-linking agent in the gel.
Suitable crosslinking agents include compounds whose molecule has a
plurality of reactive groups. Such molecular crosslinking agents
may be N,N'-methylene-bis acrylamide or divinylbenzene (DVB),
ethylene glycol dimethacrylate, divinyl ketone, vinyl methacrylate
and divinyl oxalate. Ionic crosslinkage which uses ions such as
metallic ions may also be employed. Crosslinkage using
electromagnetic waves such as gamma rays is also possible.
Cross-linking can also be based on electrostatic interactions,
hydrogen boding, hydrophobic interactions or (micro)crystal
formation.
Ionically cross-linkable polymers can be anionic or cationic in
nature and include but not limited to carboxylic, sulfate, hydroxyl
and amine functionalized polymers. The cross-linking ions used to
crosslink the polymers can be anions or cations depending on
whether the polymer is anionically or cationically cross-linkable.
Appropriate cross-linking ions include but not limited to cations
selected from the group consisting of calcium, magnesium, barium,
strontium, boron, beryllium, aluminum, iron, copper, cobalt, lead
and silver ions. Anions can be selected from but not limited to the
group consisting of phosphate, citrate, borate, succinate, maleate,
adipate and oxalate ions. More broadly, the anions are derived from
polybasic organic or inorganic acids. In some embodiments, the
cross-linking cations are calcium, iron, and barium ions. In other
embodiments, the cross-linking anion is phosphate. Cross-linking
can be carried out by contacting the polymers with a nebulized
droplet containing dissolved ions. One of ordinary skill in the art
will be able to select appropriate cross-linking agent for the
respective hydrogel used in the making of a multi-layer TE
construct. For example, the gelation of collagen or alginate occurs
in the presence of ionic cross-linker or divalent cations such as
Ca.sup.2+, Ba.sup.2+ and Sr.sup.2+.
In some embodiments, the gel comprises a biodegradable polymer
selected from the group consisting of polyanhydrides,
polyhydroxybutyric acid, polyorthoesters, polysiloxanes,
polycaprolactone, poly(lactic-co-glycolic acid), poly(lactic acid),
poly(glycolic acid), and copolymers prepared from the monomers of
these polymers.
Suitable polymers which can be used in the composition suitable for
use in the methods of the present invention include but are not
limited to one or a mixture of polymers selected from the group
consisting of polyurethanes, glycosaminoglycan, silk, fibrin,
MATRIGEL.RTM., poly-ethyleneglycol (PEG), polyhydroxy ethyl
methacrylate, polyvinyl alcohol, polyacrylamide, poly (N-vinyl
pyrolidone), poly glycolic acid (PGA), poly lactic-co-glycolic acid
(PLGA), poly e-carpolactone (PCL), polyethylene oxide, poly
propylene fumarate (PPF), poly acrylic acid (PAA), hydrolysed
polyacrylonitrile, polymethacrylic acid, polyethylene amine,
alginic acid, pectinic acid, carboxy methyl cellulose, hyaluronic
acid, heparin, heparin sulfate, chitosan, carboxymethyl chitosan,
chitin, pullulan, gellan, xanthan, collagen, gelatin, carboxymethyl
starch, carboxymethyl dextran, chondroitin sulfate, cationic guar,
cationic starch as well as salts and esters thereof. Polymers
listed above which are not ionically cross-linkable are used in
blends with polymers which are ionically cross-linkable.
Other preferred polymers include esters of alginic, pectinic or
hyaluronic acid and C2 to C4 polyalkylene glycols, e.g. propylene
glycol, as well as blends containing 1 to 99 wt % of alginic,
pectinic or hyaluronic acid with 99 to 1 wt % polyacrylic acid,
polymethacrylic acid or polyvinylalcohol. Preferred blends comprise
alginic acid and polyvinylalcohol. Examples of mixtures include but
are not limited to a blend of polyvinyl alcohol (PVA) and sodium
alginate and propyleneglycol alginate.
In some embodiments, the gel is alginate, collagen, or agarose.
As used herein, the term "magnetic material" refers to a material
or substance that is influenced by a magnetic field, i.e. relative
permeability (.mu.r) of the material is greater than unity. Such
magnetic materials are intended to include those which are referred
to as ferromagnetic, ferromagnetic, diamagnetic, paramagnetic, and
superparamagnetic. As is the conventional understanding given that
term, superparamagnetic materials exhibit magnetic properties only
when in an externally applied magnetic field, and otherwise exhibit
essentially no magnetic properties; and their total magnetism is
greater than the sum of that of the individual particles considered
separately. If the particle size of the magnetic material is
sufficiently small, the magnetic material will most likely be
superparamagnetic.
The magnetic properties of the composition are greatly influenced
by the saturation magnetization, size, and concentration of
magnetic material, as well as the strength of the external magnetic
field.
The magnetic material can be any molecule, composition, particle,
or substance, that exhibits magnetic properties when incorporated
into the matrix. The magnetic materials can be selected from the
group of elements having atomic numbers 21-29, 42, 44, and 57-70,
elements having atomic numbers 24-29 or 62-69 being especially
preferred. Preferably, a magnetic material is selected from the
group including but not limited to, rare earth metals (such as
gadolinium, terbium, dysprosium, holmium, erbium and europium),
transient metals (such as iron, nickel, cobalt, magnesium chromium
and copper), noble metals (such as rhodium, palladium), their
oxides, compositions, combinations, solid dispersions, and
alloys.
In some embodiments, the magnetic material is an iron oxide
particle. In some embodiments, the magnetic material is selected
from the group consisting of maghemite (Fe.sub.2O.sub.3), magnetite
(Fe.sub.3O.sub.4), strontium ferrite, samarium-cobalt,
neodymium-iron-boron (NIB), lodestone, pyrrhotite,
BaFe.sub.12O.sub.19, Alnico magnet alloy, transfer salts of
decamethylmetallocenes with 7,7,8,8-tetracyano-p-quinodimethane
(TCNQ) or tetracyanoethenide (TCNE) (such as
[Fe(Cp*).sub.2]+[TCNE]-, [Fe(Cp*).sub.2]+[TCNQ]-,
[Cr(Cp*).sub.2]+[TCNE]-, [Cr(Cp*).sub.2]+[TCNQ]-,
[Mn(Cp*).sub.2]+[TCNE]-, and [Mn(Cp*).sub.2]+[TCNQ]-),
hexylammonium trichlorocuprate(II)
(CuCl.sub.3(C.sub.6H.sub.11NH.sub.3), Fe based amorphous magnetic
powders, and combinations thereof. In some embodiments, the
composition of the invention comprises two or more, e.g., two,
three, four, or five, different magnetic materials.
Exemplary Fe based amorphous magnetic powders are described in U.S.
Pat. App. Pub. No. 2009/0232693, the entire contents of which are
incorporated herein by reference.
In some embodiments, magnetic material is a particle, e.g., a
magnetic nanoparticle or magnetic microparticle. Depending on the
size, porosity or pore size of matrix, magnetic particles can range
in diameter from 1 nm to 1000 .mu.m. Preferably magnetic particles
are about 1 nm to 500 nm in diameter. In some embodiments, the
magnetic particle is a magnetic nano-particle of diameter about 300
nm. Magnetic nanoparticles are a class of nanoparticle which can be
manipulated using magnetic field. Such particles commonly consist
of magnetic elements such as iron, nickel and cobalt and their
chemical compounds. Magnetic nanoparticles are well known and
methods described in the art, for example in U.S. Pat. Nos.
6,878,445; 5,543,158; 5,578,325; 6,676,729; 6,045,925 and
7,462,446, and U.S. Pat. Pub. Nos.: 2005/0025971; 2005/0200438;
2005/0201941; 2005/0271745; 2006/0228551; 2006/0233712;
2007/01666232 and 2007/0264199, the entire contents of all of which
are incorporated herein by reference.
The magnetic material should be sufficiently immobilized in the
matrix so that during any application of a magnetic field it cannot
be removed therefrom by dissolution or chemical reaction that would
be encountered, even as a result of a change in the porosity, pore
size, pore connectivity, swelling agent concentration, and/or
specific volume of the matrix. Thus, the magnetic material can be
simply physically entrapped within the matrix, or it can be
chemically bound into the matrix or complexed, encased in, or
physically immobilized by an intermediate ligand which is in turn,
chemically bound into the matrix. It is to be understood that
physical immobilization includes chelation.
In some embodiments, the magnetic material is distributed
homogeneously within the matrix material. In other embodiments, the
magnetic material is distributed heterogeneously within the matrix
material. The distribution of the magnetic material within the
matrix material can be formed by application of a magnetic field
during polymerization of the matrix material. In some embodiment,
the magnetic material is distributed within the matrix material
during polymerization of the matrix material in the presence of a
uniform magnetic field. In other embodiments, the magnetic material
is distributed within the matrix material during polymerization of
the matrix material in the presence of non-uniform magnetic field.
In some embodiments, the magnetic material is distributed into a
separate compartment within the matrix material. In some
embodiments, the magnetic material is distributed at one side
within the matrix material distant from the electromagnetic
signal.
Magnetic material-based compression compositions suitable for use
in the methods of the present invention can be prepared using
methods known in the art and easily adapted by one of skill in the
art. For example, magnetic material can be bound to the matrix by
carrying out the polymerization which forms the matrix in the
presence of chelate-forming groups and then reacting this
intermediate with an excess of the magnetic material in an aqueous
solution. If desired, a bridging group, e.g., a linker, can be
introduced between the chelate-forming groups and the matrix
backbone.
Alternatively, the magnetic material can be present in cavities
within the matrix, in the form of an insoluble or sparingly soluble
substance or composition. The incorporation of the magnetic
material within the matrix can be achieved in several ways.
In one method, dry or incompletely swollen matrix may be swelled in
an appropriate solution comprising a salt of a metal, for instance
chloride and/or sulfate of the metal, whereafter the matrix is
dried. The matrix is then swelled again in a solution, of a
substance which is capable of precipitating the metal in the form
of an insoluble or sparingly soluble magnetic material, compound or
complex. For instance the precipitating substance may be a soluble
phosphate, such as sodium phosphate, when the phosphate of the
metal is insoluble or sparingly soluble in the medium in which the
matrix is swelled. Alternatively, the precipitating substance may
be an alkali metal hydroxide when the hydroxide of the metal is
insoluble or sparingly soluble in the medium in which the matrix is
swelled.
As used herein, the term "magnetic metal" refers to any metal that
exhibits magnetic properties when it is incorporated in the matrix.
The magnetic metal may or may not exhibit magnetic properties while
it is not incorporated in the matrix. As used herein, the term
"magnetic metals" includes ions, salts, oxides or nitrides of the
metal.
In another method, dry or incompletely swollen matrix material may
be swelled in a solution comprising a solvent in which the matrix
material swells, e.g., water or dimethylsulfoxide, and a magnetic
material in a suitable chemical form and, optionally, one or more
reagents. The one or more reagents, optionally in contact with the
matrix, may produce a magnetic material in an elemental state or in
an insoluble or sparing soluble state by a chemical reaction (which
may involve the matrix), for example, a redox process, wherein the
magnetic material is finely dispersed in the matrix.
According to another method, the matrix is prepared by a process
involving a cross-linking reaction carried out in a medium in which
magnetic material or a complex thereof is dispersed, the magnetic
material or complex being insoluble or sparingly soluble in the
medium. Thus, the magnetic material or complex will become
entrapped in a dispersed form in cavities formed in the
three-dimensional network of the matrix. Where the magnetic
material is incorporated as a complex, this is preferably a chelate
complex which is insoluble or sparingly soluble in aqueous
media.
Methods for preparing ferrogels have been described, for example,
in Sahiner, N. Colloid Polym Sci (2006) 285: 283; Sauzeddle, F.
Elaissari, A. and Picho, C. Colloid Polym Sci (1999) 277: 846; Gu,
S. Shiratori, T. and Konno, M. Colloid Polym Sci (2003) 281: 1076;
Zhang, J. et al., Adv Mater (2002) 14: 1756; and Caykara, T. Yorok,
D. and Demirci, S. J App Polym Sci (2009) 112: 800, the entire
contents of all of which are incorporated herein by reference.
Once the matrix comprising magnetic material has been prepared,
macropores can be introduced by freezing the matrix at a
temperature ranging from about -10.degree. C. to about -180.degree.
C. Pores can also be created by freezing the magnetic solution
during the crosslinking reaction to create a cryogel. In some
embodiments, the matrix is frozen at a temperature of about
-15.degree. C. to about -25.degree. C. To prepare compositions
comprising pores of a size of about 700 .mu.m, a freezing
temperature of about -20.degree. C. is used. The lyophilized
composition is then swelled in the appropriate swelling agent.
Ferrogels frozen at about -20.degree. C. with pores of a size of
about 700 .mu.m have a height of about 15 mm and a diameter of
about 20 mm.
Compositions comprising different pore sizes can also be prepared.
For example, compositions comprising pores of two different sizes
for use in animal models can be prepared at a freezing temperature
of about -20.degree. C. At the freezing temperature of -20.degree.
C., two regions with different pore sizes can be produced in the
same composition, wherein the regions rich in magnetic materials,
e.g., iron oxide, have a pore size of about 340 .mu.m, and the
magnetic material-low regions have a pore size of about 140 .mu.m.
Ferrogels frozen at about -20.degree. C. with two regions of
different pore sizes, e.g., 340 .mu.m and 140 .mu.m, have a height
of about 2 mm and a diameter of about 8 mm.
Similarly, this method of pore creation is suitable for generation
of compositions comprising different pore sizes for use in humans,
wherein the resulting pores in both magnetic material-rich and -low
regions will have relatively larger pores when compared to the
composition used in animal models. Generally, diameter of a pore is
such as to allow free flow of swelling agent and/or a solvent
through the pores. Typically, pore diameters of compositions
described herein can range from about 20 .mu.m to 10000 m. In some
embodiments, the composition suitable for use in the methods of the
present invention comprises pores having mean pore diameter in
range from about 150 .mu.m to 1000 .mu.m, 200 .mu.m to 1000 .mu.m,
300 .mu.m to 1000 .mu.m, 400 .mu.m to 1000 .mu.m, 500 .mu.m to 1000
.mu.m, 600 .mu.m to 1000 .mu.m, 1000 .mu.m to 10000 .mu.m, 2000
.mu.m to 10000 .mu.m, 3000 .mu.m to 10000 .mu.m, 4000 .mu.m to
10000 .mu.m, 5000 .mu.m to 10000 .mu.m, or 6000 .mu.m to 10000
.mu.m. In some embodiments, the composition suitable for use in the
methods of the present invention comprises pores having mean pore
diameter in range from about 150 .mu.m to 750 rm. In some
embodiments, the composition comprises pores having mean pore
diameter of 200-750 .mu.m. In another embodiment, the composition
comprises pores having mean pore diameter of 250-700 .mu.m. In some
embodiments, the composition comprises pores having mean pore
diameter of 500-700 .mu.m. In other embodiments, the composition
comprises pores having mean pore diameter of 1000-7000 .mu.m. In
some embodiments, the composition comprises pores having mean pore
diameter of 2000-7000 .mu.m. In other embodiments, the composition
comprises pores having mean pore diameter of 3000-7000 .mu.m. In
some embodiments, the composition comprises pores having mean pore
diameter of 5000-7000 .mu.m. In other embodiments, the composition
comprises pores having mean pore diameter of about 100 .mu.m, 200
.mu.m, 300 .mu.m, 400 .mu.m, 500 .mu.m, 600 .mu.m, 700 .mu.m, 800
.mu.m, 900 .mu.m, 1000 .mu.m, 2000 .mu.m, 3000 .mu.m, 4000 .mu.m,
5000 .mu.m, 6000 .mu.m, 7000 .mu.m, 8000 .mu.m, 9000 .mu.m or 10000
.mu.m.
Without wishing to be bound by theory, the choice of freezing
temperature, device size and/or device shape affects porosity and
pore size of the resultant composition.
In some embodiments, the composition of the invention has an
elastic modulus in the range between 10.sup.-3 and 10.sup.3 kPa. As
used herein, the term "elastic modulus" refers to an object or
substance's tendency to be deformed elastically (i.e.,
non-permanently) when a force is applied to it. Generally, the
elastic modulus of an object is defined as the slope of its
stress-strain curve in the elastic deformation region. Specifying
how stress and strain are to be measured, including directions,
allows for many types of elastic moduli to be defined. Young's
modulus (E) describes tensile elasticity, or the tendency of an
object to deform along an axis when opposing forces are applied
along that axis; it is defined as the ratio of tensile stress to
tensile strain. It is often referred to simply as the elastic
modulus. The shear modulus or modulus of rigidity (G or .mu.)
describes an object's tendency to shear (the deformation of shape
at constant volume) when acted upon by opposing forces; it is
defined as shear stress over shear strain. The shear modulus is
part of the derivation of viscosity. The bulk modulus (K) describes
volumetric elasticity, or the tendency of an object to deform in
all directions when uniformly loaded in all directions; it is
defined as volumetric stress over volumetric strain, and is the
inverse of compressibility. The bulk modulus is an extension of
Young's modulus to three dimensions. Three other elastic moduli are
Poisson's ratio, Lame's first parameter, and P-wave modulus.
In some embodiments, the compositions suitable for use in the
methods of the present invention further comprise a compound to be
delivered to the target tissue. The compound is selected from the
group consisting of small organic or inorganic molecules;
saccharines; oligosaccharides; polysaccharides; peptides; proteins;
peptide analogs and derivatives; peptidomimetics; nucleic acids;
nucleic acid analogs and derivatives; an extract made from
biological materials such as bacteria, plants, fungi, or animal
cells; animal tissues; naturally occurring or synthetic
compositions; and any combinations thereof.
In some embodiments, the composition may further include a
bioactive agent. As used herein, "bioactive agents" or "bioactive
materials" refer to naturally occurring biological materials, for
example, extracellular matrix materials such as fibronectin,
vitronection, and laminin; cytokines; growth factors; antibodies;
vaccines and differentiation factors. "Bioactive agents" also refer
to artificially synthesized materials, molecules or compounds that
have a biological effect on a biological cell, tissue or organ.
Suitable growth factors and cytokines include, but are not limited,
to stem cell factor (SCF), granulocyte-colony stimulating factor
(G-CSF), granulocyte-macrophage stimulating factor (GM-CSF),
stromal cell-derived factor-1, steel factor, VEGF, TGF.beta.,
platelet derived growth factor (PDGF), angiopoeitins (Ang),
epidermal growth factor (EGF), bFGF, HNF, NGF, bone morphogenic
protein (BMP), fibroblast growth factor (FGF), hepatocye growth
factor, insulin-like growth factor (IGF-1), interleukin (IL)-3,
IL-1.alpha., IL-1.beta., IL-6, IL-7, IL-8, IL-11, and IL-13,
colony-stimulating factors, thrombopoietin, erythropoietin,
fit3-ligand, and tumor necrosis factor .alpha. (TNF.alpha.). Other
examples are described in Dijke et al., Bio/Technology, 7:793-798
(1989); Mulder G D, Haberer P A, Jeter K F, eds. Clinicians' Pocket
Guide to Chronic Wound Repair. 4th ed. Springhouse, Pa.:
Springhouse Corporation; 1998:85; Ziegler T. R., Pierce, G. F., and
Herndon, D. N., 1997, International Symposium on Growth Factors and
Wound Healing: Basic Science & Potential Clinical Applications
(Boston, 1995, Serono Symposia USA), Publisher: Springer
Verlag.
In some embodiments, suitable bioactive agents include but are not
limited to therapeutic agents. As used herein, the term
"therapeutic agent" refers to a substance used in the diagnosis,
treatment, or prevention of a disease. Any therapeutic agent known
to those of ordinary skill in the art to be of benefit in the
diagnosis, treatment or prevention of a disease is contemplated as
a therapeutic agent in the context of the present invention.
Therapeutic agents include pharmaceutically active compounds,
hormones, growth factors, enzymes, DNA, plasmid DNA, RNA, siRNA,
viruses, proteins, lipids, pro-inflammatory molecules, antibodies,
antibiotics, anti-inflammatory agents, anti-sense nucleotides and
transforming nucleic acids or combinations thereof. Any of the
therapeutic agents may be combined to the extent such combination
is biologically compatible.
Exemplary therapeutic agents include, but are not limited to, those
found in Harrison's Principles of Internal Medicine, 13th Edition,
Eds. T. R. Harrison et al. McGraw-Hill N.Y., N.Y.; Physicians Desk
Reference, 50th Edition, 1997, Oradell N.J., Medical Economics Co.;
Pharmacological Basis of Therapeutics, 8th Edition, Goodman and
Gilman, 1990; United States Pharmacopeia, The National Formulary,
USP XII NF XVII, 1990; current edition of Goodman and Oilman's The
Pharmacological Basis of Therapeutics; and current edition of The
Merck Index, the entire contents of all of which are incorporated
herein by reference.
Examples of therapeutic agents which may be incorporated in the
composition, include but are not limited to, narcotic analgesic
drugs; corticosteroids; hormones; pharmaceuticals for arthritis
treatment; antibiotics, including tetracyclines, penicillin,
streptomycin and aureomycin; antihelmintic; canine distemper drugs
applied to domestic animals and large cattle, for example,
phenothiazine; antitumor drugs; anti-hypertensive drugs;
anti-inflammatory agents; neuromuscular relaxants; anti-disrhythmic
drugs; vasodilating drugs; anti-hypertensive diuretics;
anticoagulants; hormones and peptides. It is understood that above
list is not full and simply represents the wide diversification of
therapeutic agents that may be included in the compositions. In
some embodiments, the therapeutic agent is Mitoxantrone, protein
(e.g. VEGF) or plasmid DNA.
The amount of therapeutic agent distributed in a composition
depends on various factors including, for example, specific agent;
function which it should carry out; required period of time for
release of a the agent; quantity to be administered. Generally,
dosage of a therapeutic agent i.e. amount of therapeutic agent in
composition, is selected from the range about from 0.001% (w/w) up
to 95% (w/w), preferably, from about 5% (w/w) to about 75% (w/w),
and, most preferably, from about 10% (w/w) to about 60% (w/w).
In some embodiments, the composition comprises a cell, e.g. a
biological cell. One way to incorporate cells into the composition
is by re-swelling a dried or partially dried composition of the
invention in an aqueous solution comprising the cells to be
incorporated. The aqueous solution can comprise enough cells, such
that from about 10.sup.4 to about 10.sup.8 cells/ml are deposited
in the composition. In some embodiments, the aqueous solution
comprises from about 10.sup.4 to about 10.sup.6 cells/ml. In one
embodiment, the aqueous solution comprises about 5.times.10.sup.5
cells/ml.
In some embodiments, the composition comprises more that one cell
type. This can be accomplished by having two or more different cell
types in aqueous solution used for swelling. When two or more
different cell types are to be incorporated into the composition,
total number of cells in the aqueous solution ranges from about
10.sup.4 to about 10.sup.8 cells/ml, about 10.sup.4 to about
10.sup.6 cells/ml, or about 10.sup.5 cells/ml.
Cells amenable to be incorporated into the composition include, but
are not limited to, stem cells (embryonic stem cells, mesenchymal
stem cells, bone-marrow derived stem cells and hematopoietic stem
cells), chrondrocytcs progenitor cells, pancreatic progenitor
cells, myoblasts, fibroblasts, keratinocytes, neuronal cells, glial
cells, astrocytes, pre-adipocytes, adipocytes, vascular endothelial
cells, hair follicular stem cells, endothelial progenitor cells,
mesenchymal cells, neural stem cells and smooth muscle progenitor
cells.
In some embodiments, the cell is a genetically modified cell. A
cell can be genetically modified to express and secrete a desired
compound, e.g., a bioactive agent, a growth factor, differentiation
factor, cytokines, and the like. Methods of genetically modifying
cells for expressing and secreting compounds of interest are known
in the art and easily adaptable by one of skill in the art.
Differentiated cells that have been reprogrammed into stem cells
can also be used. For example, human skin cells reprogrammed into
embryonic stem cells by the transduction of Oct3/4, Sox2, c-Myc and
Klf4 (Junying Yu, et. al., 2007, Science 318: 1917-1920; Takahashi
K. et. al., 2007, Cell 131: 1-12).
Cells useful for incorporation into the composition can come from
any source, for example human, rat or mouse. Human cells include,
but are not limited to, human cardiac myocytes-adult (HCMa), human
dermal fibroblasts-fetal (HDF-f), human epidermal keratinocytes
(HEK), human mesenchymal stem cells-bone marrow, human umbilical
mesenchymal stem cells, human hair follicular inner root sheath
cells, human umbilical vein endothelial cells (HUVEC), and human
umbilical vein smooth muscle cells (HUVSMC), human endothelial
progenitor cells, human myoblasts, human capillary endothelial
cells, and human neural stem cells.
Exemplary rat and mouse cells include, but not limited to, RN-h
(rat neurons-hippocampal), RN-c (rat neurons-cortical), RA (rat
astrocytes), rat dorsal root ganglion cells, rat neuroprogenitor
cells, mouse embryonic stein cells (mESC) mouse neural precursor
cells, mouse pancreatic progenitor cells mouse mesenchymal cells
and mouse endodermal cells.
In some embodiments, tissue culture cell lines can be used in the
compositions described herein. Examples of cell lines include but
are not limited to C166 cells (embryonic day 12 mouse yolk), C6
glioma Cell line, HL1 (cardiac muscle cell line), AML12
(nontransforming hepatocytes), HeLa cells (cervical cancer cell
line) and Chinese Hamster Ovary cells (CHO cells).
An ordinary skill artisan in the art can locate, isolate and expand
such cells. In addition, the basic principles of cell culture and
methods of locating, isolation and expansion and preparing cells
for tissue engineering are described in "Culture of Cells for
Tissue Engineering" Editor(s): Gordana Vunjak-Novakovic, R. Ian
Freshney, 2006 John Wiley & Sons, Inc., and in "Cells for
tissue engineering" by Heath C. A. (Trends in Biotechnology, 2000,
18:17-19) and the entire contents of all of which are incorporated
herein by reference.
The bioactive agent can be covalently linked to the matrix through
a linker. The linker can be a cleavable linker or non-cleavable
linker, depending on the application. As used herein, a "cleavable
linker" refers to linkers that are capable of cleavage under
various conditions. Conditions suitable for cleavage can include,
but are not limited to, pH, UV irradiation, enzymatic activity,
temperature, hydrolysis, elimination and substitution reactions,
redox reactions, and thermodynamic properties of the linkage. In
many cases, the intended nature of the conjugation or coupling
interaction, or the desired biological effect, will determine the
choice of linker group.
In some embodiments, the bioactive agent is bound to the matrix by
a hydrolyzable bond. In some embodiments, the cell or the bioactive
agent has a mean free path in the composition that is shorter than
the mean free path of the cell or the bioactive agent in water.
A composition suitable for use in the methods of the present
invention described herein can be administered to a subject by any
appropriate route known in the art. As used herein, the term
"administer" refers to the placement of a composition into or onto
a subject by a method or route which results in at least partial
localization of the composition at a desired site such that a
desired effect is produced.
Exemplary modes of administration include, but are not limited to,
injection, implantation, or topical application such as placing the
compositions over the skin. "Injection" includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intraventricular, intracapsular, intraorbital, intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous,
subcuticular, intraarticular, sub capsular, subarachnoid,
intraspinal, intracerebro spinal, and intrasternal injection and
infusion. In some embodiments, the compositions are administered by
intravenous infusion or injection. In some embodiments, the
compositions are injected at the site of tissue damage. In other
embodiments, the compositions are implanted at the site of injury
upon major surgery to assist tissue regeneration or to treat tissue
injury.
Compositions that are to be implanted can additionally include one
or more additives. Additives may be resolving (biodegradable)
polymers, mannitol, starch sugar, inosite, sorbitol, glucose,
lactose, saccharose, sodium chloride, calcium chloride, amino
acids, magnesium chloride, citric acid, acetic acid,
hydroxyl-butanedioic acid, phosphoric acid, glucuronic acid,
gluconic acid, poly-sorbitol, sodium acetate, sodium citrate,
sodium phosphate, zinc stearate, aluminium stearate, magnesium
stearate, sodium carbonate, sodium bicarbonate, sodium hydroxide,
polyvinylpyrolidones, polyethylene glycols, carboxymethyl
celluloses, methyl celluloses, starch or their mixtures.
For administration to a subject, composition comprising a bioactive
agent can be formulated together with one or more pharmaceutically
acceptable carriers (additives) and/or diluents. As used herein,
the term "pharmaceutically acceptable" refers to those compounds,
materials, compositions, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
As used herein, the term "pharmaceutically-acceptable carrier"
means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc
stearate, or steric acid), or solvent encapsulating material,
involved in carrying or transporting a bioactive agent from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can
serve as pharmaceutically-acceptable carriers include: (1) sugars,
such as lactose, glucose and sucrose; (2) starches, such as corn
starch and potato starch; (3) cellulose, and its derivatives, such
as sodium carboxymethyl cellulose, methylcellulose, ethyl
cellulose, microcrystalline cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents,
such as magnesium stearate, sodium lauryl sulfate and talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl
oleate and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters,
polycarbonates and/or polyanhydrides; (22) bulking agents, such as
polypeptides and amino acids (23) serum component, such as serum
albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and
(23) other non-toxic compatible substances employed in
pharmaceutical formulations. Wetting agents, coloring agents,
release agents, coating agents, sweetening agents, flavoring
agents, perfuming agents, preservative and antioxidants can also be
present in the formulation. The terms such as "excipient",
"carrier", "pharmaceutically acceptable carrier" or the like are
used interchangeably herein.
The composition suitable for use in the methods of the present
invention can be of rectangular form from about 0.5 to about 50 cm
in length and from about 0.1 to about 10 cm in width. For example,
the composition may have a length from about 1 to about 10 cm,
about 10 to about 50 cm, about 1 to about 20 cm, about 1 to about
30 cm, about 1 to about 40 cm, about 5 to about 20 cm, about 5 to
about 30 cm, about 5 to about 40 cm, about 10 to about 30 cm, or
about 10 to about 40 cm. The composition may have a width from
about 0.1 to about 1 cm, 1 to about 10 cm, about 2 to about 10 cm,
about 3 to about 10 cm, about 4 to about 10 cm, about 5 to about 10
cm, or about 6 to about 10 cm.
The composition suitable for use in the methods of the present
invention can also be of cylindrical form from about 0.5 to about
30 cm in diameter and from about 0.1 to about 10 cm in length. For
example, the composition may have a diameter from about 0.5 to
about 1 cm, about 1 to about 10 cm, about 1 to about 20 cm, about 1
to about 30 cm, about 1 to about 5 cm, about 5 to about 10 cm,
about 5 to about 20 cm, about 5 to about 30 cm, about 10 to about
30 cm, or about 20 to about 30. The composition may have a length
from about 0.1 to about 1 cm, about 1 to about 10 cm, about 2 to
about 10 cm, about 3 to about 10 cm, about 4 to about 10 cm, about
5 to about 10 cm, or about 6 to about 10 cm.
The composition suitable for use in the methods of the present
invention can further be of circular shape with a diameter from
about 0.5 to about 30 cm. For example, the composition may have a
diameter from about 0.1 to about 10 cm, about 10 to about 30 cm,
about 1 to about 20 cm, about 1 to about 30 cm, about 5 to about 10
cm, about 5 to about 20 cm, about 5 to about 30 cm, about 10 to
about 20 cm, or about 20 to about 30 cm.
In some cases, the composition can be of a spherical shape. When
the composition is in a spherical shape, its diameter can range
from about 0.5 to about 10 cm. In some embodiments, the diameter of
the composition is from about 1 to about 10 cm. In other
embodiments, the diameter of the composition is from about 1 to
about 5 cm. In some embodiments, the diameter of the composition is
from about 5 to about 10 cm. In other embodiments, the diameter of
the composition is from about 1 to about 3 cm. In some embodiments,
the diameter of the composition is from about 3 to about 5 cm. In
other embodiments, the diameter of the composition is from about 3
to about 10 cm.
B. Controller-Based Compression Devices
A controller-based compression device suitable for use in the
methods of the present invention comprises a surrounding member
configured to encircle a body part including a site of tissue
damage and apply cyclic mechanical compressions to the tissue, and
a controller configured to generate the cyclic mechanical
compressions in the surrounding member.
In some embodiments, the surrounding member includes a sleeve that
encircles a body part that includes the site of tissue damage. In
some embodiments, the compression device is configured to
externally encircle a body part or at least a portion of a body
part that includes the site of tissue damage. In some embodiments,
the compression device is configured to be disposed at least
partially internally within the body and at least partially
encircle the body that includes the site of tissue damage.
In some embodiments, a controller associated with the surrounding
member is used to generate the cyclic mechanical compressions in
the surrounding member. As used herein, a "controller" is a device
or system configured to generate cyclic mechanical compressions and
to control properties of the cyclic mechanical compressions, such
as, the frequency, the amplitude and/or the duration of the cyclic
mechanical compressions being generated. In some embodiments, the
controller includes a microcontroller that controls the properties
of the cyclic mechanical compressions. In some embodiments, the
controller includes storage for holding machine readable
instructions, measurements of pressure from a pressure sensor, or
other information. In some embodiments, microcontroller includes
storage for holding machine readable instructions, measurements of
pressure from a pressure sensor, or other information. In some
embodiments, the controller includes additional storage that is not
included in the microcontroller, but is accessible to the
microcontroller for holding machine readable instructions,
measurements of pressure from a pressure sensor, or other
information.
In some embodiments, the controller is included in the compression
device. For example, the controller and the surrounding member may
be included in a single unit in the compression device. In some
embodiments, the compression device includes the controller and the
surrounding member and is configured to be worn. In some
embodiments, a compression device suitable for use in the methods
of the present invention described herein is suitable for wearable
applications. In some embodiments, the compression device is
wearable and untethered such that the wearer can be mobile during
the cyclic mechanical compressions. In some embodiments, the
controller is separate from the compression device. In some
embodiments, the controller is separate from, but coupleable with
one or more components of a compression device (e.g., coupleable
with a surrounding member of the compression device). In some
embodiments, the controller is portable.
In some embodiments, the compression device is electromagnetically
actuated, pneumatically actuated or hydraulically actuated to apply
the cyclic mechanical compressions to the tissue. In some
embodiments, the controller includes a pump. In some embodiments,
the pump is a pneumatic air pump. In some embodiments the
controller includes a microcontroller that provides instructions to
the pump.
In some embodiments, the surrounding member includes one or more
inflatable portions (e.g., one or more inflatable chambers, one or
more inflatable bladders, one or more inflatable members, or a
combination of the aforementioned). In some embodiments, a
surrounding member of the compression device comprises at least 2,
3, 4, 5, 6, 7, 8, 9, or 10 inflatable portions. In some
embodiments, the one or more inflatable portions are configured to
be inflated with a gas, e.g., air. In other embodiments, the one or
more inflatable portions are configured to be inflated with a
liquid, e.g., water. In some embodiments, the one or more
inflatable portions are configured to be inflated by an
electromagnetic signal. In some embodiments, the one or more
inflatable portions are configured to be independently inflated and
deflated. In some embodiments, only some of the one or more
inflatable portions are configured to be independently inflated and
deflated.
In some embodiments, at least partial inflation of at least some of
the one or more inflatable portions is used to apply the cyclic
mechanical compressions to the tissue. In some embodiments,
increasing a pressure of inflation of at least some of the one or
more inflatable portions is used to apply the cyclic mechanical
compressions to the tissue. In some embodiments, at least some of
the one or more inflatable portions aid in contacting the tissue
with the compression device (e.g., to aid in fitting the
surrounding member to the body part). In some embodiments, some of
the one or more inflatable portions aid in contacting the tissue
with the compression device and others of the inflatable portions
are used to apply the cyclic mechanical compressions to the tissue.
For example, some inflatable portions are used to maintain a
constant low level of inflation to maintain a fit of the
surrounding member around a body part while one or more other
inflatable portions disposed over the site of tissue damage are
cyclically inflated and deflated.
In some embodiments, at least some of the one or more inflatable
portions are parts of a unitary structure (e.g., different
inflatable portions of a unitary sleeve that may be independently
inflatable). In some embodiments, at least some of the one or more
inflatable portions are separate inflatable members that are not
portions of a unitary structure (e.g., a balloon or bladder
disposed within a pocket of a sleeve that includes inflatable
sections).
Inflation of some or all of the inflatable portions of the
surrounding member may occur sequentially or simultaneously, or in
part sequentially and in part simultaneously, to generate cyclic
mechanic compression at a target site, e.g., the site of tissue
damage, thereby promoting tissue regeneration and/or repair at the
site of tissue damage. Inflation of at least some of the one or
more inflatable portions can be regulated by the controller of the
compression device. For example, the controller (e.g., a
microcontroller system) can be programmed to control one or more
valves associated individually, groupwise, or collectively, with
the one or more inflatable portions. For example, each inflatable
portion may have an associated valve to control flow, one valve may
control a flow into a group of inflatable portions while another
valve may control flow into another group of inflatable portions,
or one valve may control flow into all the inflatable portions.
Using the one or more valves, the one or more inflatable portions
can be inflated, or the inflation pressure increased, for a
pre-determined time (e.g., 500 ms), followed by a period of time
(e.g., 500 ms) of deflation, when no pressure is applied or when a
decreased pressure is applied. Such a cycle of inflation, or
increased inflation pressure, and deflation may be applied at a
pre-determined frequency, e.g., 1 Hertz. In some embodiments, the
controller includes storage holding readable instructions for
applying the inflation pressure to at least some of the one or more
inflatable portions of the surrounding member to generate the
cyclic mechanical compressions in the surrounding member.
The particular time periods for inflation and deflation can vary
depending on the conditions of the subjects and/or the purpose of
the methods. In some embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, for at least 5
seconds, followed by a deflation period of 5 seconds. In other
embodiments, the inflatable portions can be inflated, or the
inflation pressure increased, for at least 10 seconds, followed by
a deflation period of at least 10 seconds. In some embodiments, the
inflatable portions can be inflated, or the inflation pressure
increased, for at least 15 seconds, followed by a deflation period
of at least 15 seconds. In other embodiments, the inflatable
portions can be inflated, or the inflation pressure increased, for
at least 20 seconds, followed by a deflation period of at least 20
seconds. In some embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, for at least 30
seconds, followed by a deflation period of at least 30 seconds. In
other embodiments, the inflatable portions can be inflated, or the
inflation pressure increased, for at least 60 seconds, followed by
a deflation period of at least 60 seconds.
In some embodiments, the inflatable portions can be inflated, or
the inflation pressure increased, for at least 1 minute every 1, 2,
3, 4, 5, 6, 7, 8, 9 or 10 hours. In other embodiments, the
inflatable portions can be inflated, or the inflation pressure
increased, for at least 2 minutes every 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10 hours. In some embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, for at least 5
minutes every 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. In other
embodiments, the inflatable portions can be inflated, or the
inflation pressure increased, for at least 10 minutes every 1, 2,
3, 4, 5, 6, 7, 8, 9 or 10 hours. In some embodiments, the
inflatable portions can be inflated, or the inflation pressure
increased, for at least 15 minutes every 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10 hours. In other embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, for at least 20
minutes every 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours.
In some embodiments, the inflatable portions can be inflated, or
the inflation pressure increased, for at least 1 minute every 12
hours. In other embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, for at least 2
minutes every 12 hours. In some embodiments, the inflatable
portions can be inflated, or the inflation pressure increased, for
at least 5 minutes every 12 hours. In other embodiments, the
inflatable portions can be inflated, or the inflation pressure
increased, for at least 10 minutes every 12 hours. In some
embodiments, the inflatable portions can be inflated, or the
inflation pressure increased, for at least 15 minutes every 12
hours. In other embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, for at least 20
minutes every 12 hours.
The frequency for applying mechanical compressions can also vary
from subject to subject, or from condition to condition. In some
embodiments, the inflatable portions can be inflated, or the
inflation pressure increased, with a frequency from at least a 1 Hz
to 500 Hz. In some embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, with at least a 1 Hz
frequency. In other embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, with at least a 2 Hz
frequency. In some embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, with at least a 3 Hz
frequency. In other embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, with at least a 4 Hz
frequency. In some embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, with at least a 5 Hz
frequency. In other embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, with at least a 6 Hz
frequency. In some embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, with at least a 7 Hz
frequency. In other embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, with at least a 8 Hz
frequency. In some embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, with at least a 9 Hz
frequency. In other embodiments, the inflatable portions can be
inflated, or the inflation pressure increased, with a frequency of
at least 10 Hz, 15 Hz, 20 Hz, 30 Hz, 40 Hz, 50 Hz, 60 Hz, 70 Hz, 80
Hz, 90 Hz, 100 Hz, 200 Hz, 300 Hz, 400 Hz or 500 Hz.
Accordingly, the capability to regulate the frequency as well as
the duration of inflation of the inflatable portions allows the
level of cyclic mechanical compression to be varied from subject to
subject. For example, a subject with superficial disease may be
treated effectively by a low level of compression whereas a subject
with a severe condition may need a higher level of compression. In
some embodiments, the compression devices are configured to exert a
peak pressure of about 1 kPa at the site of tissue damage. In other
embodiments, the compression devices are configured to exert a peak
pressure of about 2 kPa. In some embodiments, the compression
devices are configured to exert a peak pressure of about 3 kPa. In
other embodiments, the compression devices are configured to exert
a peak pressure of about 4 kPa. In certain embodiments, the
compression devices are configured to exert a peak pressure of
about 5 kPa. Thus, actuation of the compression devices results in
cyclic mechanical compressions at the site of tissue damage,
thereby promoting tissue regeneration and/or repair at the site of
tissue damage. It is possible to provide the pressure profile
needed to treat these various indications through the use of a
compression device as described herein.
The controller may further comprise at least one pressure sensor
(e.g., air pressure sensor, or surface pressure sensors that can be
resistive or capacitance measures) attached to the surrounding
member and disposed within the surrounding member (e.g., internally
within a sleeve of the surrounding member) or disposed between a
force exerting portion of the surrounding member and a body part
(e.g., on an inward facing surface of a sleeve configured to be
positioned between an inflatable portion that exerts compressive
pressure on the site of tissue damage and the body part of the
subject). The pressure sensor provides readings of the pressure
experienced by the site of tissue damage, e.g., the limb, due to
the inflation of one or more inflatable portions of the surrounding
member by the controller. These sensors may be based on conductive
fabrics or elastomers and can enable monitoring of applied pressure
to signals delivered to the one or more inflatable portions of the
surrounding member. The controller may also comprise an
accelerometer or other soft strain and pressure sensors that can
measure the frequency and amplitude of the vibration or cyclic
mechanical compressions at one or more inflatable portions in the
surrounding member.
Monitoring the actual pressure experienced by a site of tissue
damage, e.g., a limb of a subject, due to the compression device,
enables the compression device to provide a pre-determined
compression profile to the site of tissue damage, e.g., the limb.
The pre-determined compression pressure profile may be selected by
a heath case professional based on the subject's condition.
In some embodiments, the sensor also allows the compression device
to regulate, e.g., increase or decrease, pressure at a specific
location of a body part when desired to do so. In some embodiments,
for a compression device including multiple inflatable portions the
controller may be programmed to inflate only selected portions of
the compression device. For example, where there is a site of
tissue damage at a specific part of a limb of a subject, not all
the inflatable portions need be inflated to apply cyclic mechanical
compressions, and only those specific ones overlying the site of
tissue damage will be inflated.
In some embodiments, for a compression device including multiple
inflatable portions (e.g., multiple independently inflatable
chambers), the compression device includes a plurality of sensors
with each sensor associated with a corresponding inflatable portion
to monitor the pressure experienced at a site of tissue damage,
e.g., a limb of a subject, due to pressure from that inflatable
portion. In some embodiments, with a plurality of independently
inflatable portions (e.g., independently inflatable chambers), a
sensor is associated with each independently inflatable portion to
monitor the pressure experienced at a site of tissue damage due to
pressure from that independently inflatable portion. This enables
the compression device to precisely control the pressure in each
inflatable portion to achieve the desired pressure profile. In some
embodiments, the controller includes storage storing machine
readable instructions for applying an inflation pressure to one or
more inflatable portions of a surrounding member to generate the
cyclic mechanical compressions in the surrounding member, receiving
information regarding a pressure measurement from a pressure sensor
associated with one of the inflatable portions, and modifying one
or both of a level of the inflation pressure applied or a time
period that the inflation pressure is applied to the one or more
inflatable portions based on the information regarding a pressure
measurement received from the pressure sensor.
In some embodiments, due to the sensors and monitoring capacity of
the compression device and the microcontroller system present in
the controller, it is possible to monitor the usage of the
compression device by a subject. In some embodiments, the
controller includes storage configured to store information a from
a pressure sensor regarding a plurality of pressure measurements
acquired during application of cyclic mechanical compressions to
tissue of the subject. For example, the controller may store
information regarding the actual pressures applied against the
subject's tissue during cyclic compressions to aid in confirming
that the device has been used properly. Knowledge of the extent of
usage will enable a heath care professional to prescribe the most
suitable treatment for the next stage of healing or prevention.
In some embodiments, a surrounding member of a compression device
includes one or more soft actuators, which are used to apply cyclic
mechanical compressions to the tissue. As used herein, the term
"soft actuator" refers to an actuator consisting of elastomeric
matrices with embedded flexible materials (e.g., cloth, paper,
fiber, particles). Soft actuators for use in the compression
devices include any actuators known in the art. Exemplary actuators
include, but not limited to, a fiber reinforced actuator, a Pneunet
bending actuator, a McKibben actuator, a pleated air muscle, a
balloon, an inflatable, a motor, a vibrating motor, a cable, an
electroactive material, e.g., a shape memory alloy, an
electrostatic or a dielectric elastomer, and combinations thereof.
The controller can control actuation of the soft actuators through
the control of fluid (i.e., liquid or gas) flow into and out of the
soft actuators. The controller can also control actuation of the
soft actuators by applying an electromagnetic signal.
Soft actuators suitable for use in the compression devices of the
present invention can be designed and fabricated using any methods
and materials that are known in the art and easily adapted by one
of skill in the art. These actuators can be rapidly fabricated in a
multi-step molding process and can achieve combinations of
pre-designed motions, such contraction, extension, bending and
twisting with simple control inputs such as pressurized fluid or
air. See, e.g., U.S. Pat. Nos. 6,718,766, 6,772,673,
US2014/0109560A1, WO2013/130760A2, WO2015/157560A1 and
WO2015/061444A1. The entire contents of each of the foregoing
applications are incorporated herein by reference.
In some embodiments, a surrounding member includes at least 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 soft actuators. In some embodiments, an
inflatable portion of a surrounding member includes a channel in
which the soft actuator is disposed. In some embodiment, the
actuators are integrated into the surrounding member in discrete
parts. In other embodiments, the actuators are integrated together
as part of the manufacturing process.
In some embodiments, a surrounding member includes a matrix
material that encircles or surrounds a body part that includes the
site of tissue damage. Matrix materials for use in the compression
devices include, but are not limited to: a textile, a fabric, a
mesh, a silicone elastomer, a rubber, and combinations of the
aforementioned.
In some embodiments, a surrounding member of a compression device
includes a balloon (e.g., an urethane balloon in a sleeve including
the matrix material). In some embodiments, the balloon is disposed
within a pocket of the matrix material (e.g., in a pocket of a
sleeve). In other embodiments, a balloon is disposed over the
subject's skin at the site of tissue damage and the matrix material
(e.g., a sleeve) is disposed encircling the balloon and the body
part (e.g., a part of a limb) that is the site of tissue damage.
The compression device applies cyclic mechanical compressions to
the damaged tissue through the skin, thus promoting regeneration
and/or repair of the damaged tissue.
In some embodiments, the balloon of the compression device can be
of a spherical shape. When the balloon is in a spherical shape, its
diameter can range from about 0.5 to about 10 cm; about 1 to about
10 cm; about 1 to about 5 cm; about 5 to about 10 cm; about 1 to
about 3 cm; about 3 to about 5 cm or about 3 to about 10 cm.
The size and/or a shape of the surrounding member may be determined
based on the size, shape or structure of the body part that
includes the site of the tissue damage. For example, the size and
shape of a surrounding member for tissue damage in a knee may be
different than the size and shape of a surrounding member for
tissue damage in a shoulder, in a finger, or in an abdomen.
Further, the size and/or shape of the surrounding member may be
determined, at least in part, by the size and shape of an area of
tissue damage in the body part. In some embodiments, the
compression device includes one surrounding member. In some
embodiments, the compression device includes multiple surrounding
members.
In some embodiments, the surrounding members (e.g., sleeves) of the
compression devices suitable for use in the methods of the present
invention described herein are customizable for different
geometries and morphologies. The surrounding member may be
adjustable or configurable for encircling different sizes and/or
different types of body parts. In some embodiments, the surrounding
members can wrap around the site of the tissue damage (e.g., the
limb) adopting the shape of the limb. In other embodiments, a
surrounding member has a shape similar to the anatomical features
of a body part of a subject (e.g., a leg, a foot, an arm, a wrist,
a neck, a shoulder, a knee or a joint) enabling the surrounding
member to fit comfortably onto the body part of the subject.
Compression devices can be configured to surround any desired
location (e.g., a site of tissue damage) of a subject in need
thereof for application of cyclic mechanical compressions at the
target location to promote regeneration and/or repair of the
damaged tissue in the subject.
In some embodiments, the surrounding members of the compression
devices are light weight and low profile, which enables a subject
to use the compression devices wearing ordinary clothes and shoes.
In some embodiments, a compression device suitable for use in the
methods of the present invention described herein is suitable for
wearable applications. In some embodiments, the compression device
is configured to apply the cyclic mechanical compressions to the
tissue while being worn and being physically untethered to any
other medical equipment. In some embodiments, the compression
device is self-contained and configured to be worn. Accordingly, in
some embodiments, the compression devices suitable for use in the
methods of the invention are convenient to operate and are suitable
for normal routine life style.
Unlike the existing commercially available wearable (e.g.,
self-contained and untethered) devices, which primarily focus on
the use of passive compression, in some embodiments, compression
devices suitable for use in the methods of the present invention
are able to generate active mechanical compressions while being
worn and untethered. By incorporating soft actuators or inflatable
portions and controllers into the wearable compression devices of
some embodiments, active and programmable cyclic mechanical
compressions can be generated, which promote regeneration and/or
repair of tissue at the site of tissue damage and improve the
healing potential of the damaged tissue.
One of ordinary skill in the art will appreciate that some of the
methods described herein can also be performed using compression
devices and compression systems that are tethered (e.g., where not
all components of the compression device or compression system is
wearable) or that are not self-contained.
In some embodiments, the surrounding members of the compression
devices that are configured to surround a site of tissue injury are
oblong in shape (e.g., rectangular, oval, elliptical, or irregular
in shape with one dimension longer than the other) from about 0.5
to about 50 cm in length and from about 0.1 to about 10 cm in
width. For example, the surrounding members of the compression
device may have a length from about 1 to about 10 cm, about 10 to
about 50 cm, about 1 to about 20 cm, about 1 to about 30 cm, about
1 to about 40 cm, about 5 to about 20 cm, about 5 to about 30 cm,
about 5 to about 40 cm, about 10 to about 30 cm, or about 10 to
about 40 cm. The surrounding members of the compression device may
have a width from about 0.1 to about 1 cm, 1 to about 10 cm, about
2 to about 10 cm, about 3 to about 10 cm, about 4 to about 10 cm,
about 5 to about 10 cm, or about 6 to about 10 cm.
In some embodiments, the surrounding members of the compression
devices that are configured to surround at a site of injury are, at
least in part, cylindrical in shape with the cylindrical portion
being about 0.5 to about 30 cm in diameter and from about 0.1 to
about 10 cm in length. For example, a surrounding member of a
compression device may have a cylindrical portion with a diameter
of about 0.5 to about 1 cm, about 1 to about 10 cm, about 1 to
about 20 cm, about 1 to about 30 cm, about 1 to about 5 cm, about 5
to about 10 cm, about 5 to about 20 cm, about 5 to about 30 cm,
about 10 to about 30 cm, or about 20 to about 30. The cylindrical
portion may have a length from about 0.1 to about 1 cm, about 1 to
about 10 cm, about 2 to about 10 cm, about 3 to about 10 cm, about
4 to about 10 cm, about 5 to about 10 cm, or about 6 to about 10
cm.
A composition suitable for use in the methods of the present
invention described herein can be administered to a subject by any
appropriate route known in the art. Exemplary modes of
administration include, but are not limited to, implantation, or
topical application such as placing the composition over the skin.
In some embodiments, the compression device is configured to be
disposed externally to the body and surrounds at least a portion of
a body part that is the site of tissue damage (e.g., a limb, a
spine, a neck, a waist, a shoulder, a knee, a joint, an ankle, a
calf, a thigh, a foot, a hand, a wrist, an arm, a shoulder, or an
axilla). For example, the compression devices are wrapped around
the site of tissue damage or placed over the site of tissue damage
to apply cyclic mechanical compressions over the target site. In
some embodiments, the compression device is configured to be
implanted, or disposed, at least partially, internally within the
body and to, at least partially, surround a body part that includes
the site of tissue damage. In some embodiments, the surrounding
member of the compression device is implanted at the site of tissue
damage at least partially encircling or surrounding an esophagus of
a subject. In other embodiments, the surrounding member of the
compression device is implanted at the site of tissue damage
encircling or surrounding an urethral or anal sphincter of a
subject. In some embodiments, the compression devices are implanted
at the site of tissue damage upon major surgery to assist tissue
regeneration or to treat tissue damage.
Compositions that are to be implanted can additionally include one
or more additives. Additives may be resolving (biodegradable)
polymers, mannitol, starch sugar, inosite, sorbitol, glucose,
lactose, saccharose, sodium chloride, calcium chloride, amino
acids, magnesium chloride, citric acid, acetic acid,
hydroxyl-butanedioic acid, phosphoric acid, glucuronic acid,
gluconic acid, poly-sorbitol, sodium acetate, sodium citrate,
sodium phosphate, zinc stearate, aluminium stearate, magnesium
stearate, sodium carbonate, sodium bicarbonate, sodium hydroxide,
polyvinylpyrolidones, polyethylene glycols, carboxymethyl
celluloses, methyl celluloses, starch or their mixtures.
FIG. 8 schematically depicts components of a compression device 100
in accordance with some embodiments of the invention. The
compression device 100 includes a surrounding member 110 configured
to encircle a body part including a site of tissue damage and apply
cyclic mechanical compressions to the tissue at the site of tissue
damage and a controller 120 configured to generate the cyclic
mechanical compressions in the surrounding member. The surrounding
member 110 includes an inflatable portion in the form of a balloon
112. The controller 120 includes a microcontroller 122 configured
to control one or more of the following: a frequency of compression
cycles, a total duration of compression cycles, a length of a
period of increasing compression in a single cycle, a length of a
period of decreasing compression in a single cycle, or a peak
compression level. The controller 120 also includes a pump 124
configured to provide fluid in the form of a liquid or a gas to the
inflatable portion (e.g., balloon 112). The controller 120 also
includes a valve 126 configured to control fluid flow between the
pump 124 and the inflatable portion (e.g., balloon 112). Operation
of the valve 126 is controlled by the microcontroller 122.
In some embodiments, the compositions suitable for use in the
methods of the present invention described herein are free of any
biologics as described herein, e.g., a bioactive agent, or a cell.
Application of mechanical stimulation alone, i.e., cyclic
mechanical compressions, to the target tissue is sufficient to
enhance functional tissue regeneration and to reduce fibrosis and
inflammation at the injured tissue. Accordingly, the biologic-free
composition may offer a simple yet effective alternative to
cell-based or drug-based therapies when treating tissue
injuries.
In some embodiments, the compositions suitable for use in the
methods of the present invention are combined with existing
biologic-based therapies to treat tissue injury. For example,
incorporation of cyclic mechanical compressions into existing drug
and cell delivery systems, that employ both mechanical and
biological interventions, could potentially lead to a new
combinatorial therapies that generate enhanced regenerative
outcomes.
The present invention is further illustrated by the following
examples, which are not intended to be limiting in any way. The
entire contents of all references, patents and published patent
applications cited throughout this application, as well as the
Figures, are hereby incorporated herein by reference.
Examples
The following methods were used in the examples below unless
otherwise specified.
Materials and Methods
Materials
Medical grade, high molecular weight (.about.250 kDa) sodium
alginate with high guluronate content (Protanal LF 20/40) was
purchased from FMC Biopolymers (Oslo, Norway). Alginates were used
following covalent RGD modification and dialysis purification, as
previously described (Rowley et al., (1999) Biomaterials 20(1):
45-53). All other chemicals including adipic acid dihydrazide
(AAD), 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC), MES,
1-hydroxybenzotriazole (HOBT), Iron(II,III) oxide powder (<5
.mu.m, 310069) were purchased from Sigma-Aldrich (St. Louis,
Mo.).
Animals and Surgical Procedures
All animal work was performed in compliance with NIH and
institutional guidelines. Six-week-old female wild-type C57BL/6J
mice (Jackson Laboratories, Bar Harbor, Me., USA) were anesthetized
with an intraperitoneal injection of ketamine (80 mg kg.sup.-1) and
xylazine (5 mg kg.sup.-1). For myotoxin injuries, the tibialis
anterior muscles of the right legs of anesthetized mice were
injected with 10 .mu.l of 10 .mu.g/ml Notexin Np myotoxin from
Notechis Scutatus snake venom (Latoxan) using a 25 .mu.l Hamilton
syringe. Six days after notexin injection, hindlimb ischemia was
induced by unilateral external iliac and femoral artery and vein
ligation, as previously described (Silva E A & Mooney D J
(2007) J. Thromb. Haemost. 5(3):590-598; Chen R R, et al. (2007)
FASEB J. 21(14):3896-3903). After vessel ligation, a hydrated
ferrogel scaffold was placed subcutaneously on the tibialis
anterior muscle in certain conditions and the incision was
surgically closed.
Ischemia and Perfusion Analysis
Blood perfusion measurements of the ischemic and normal limb were
performed on anesthetized animals (n=10) using a Laser Doppler
Perfusion Imaging (LDPI) analyzer (PeriScan PIM II, Perimed
Instruments, Ardmore, Pa.). Entire hindlimbs were scanned under
basal conditions and then every other day following surgery.
Perfusion was calculated as the ratio of ischemic to non-ischemic
limb perfusion for each animal.
Ferrogel Scaffold Stimulation
Biphasic ferrogels (7 wt % iron oxide) were fabricated with
alginate covalently modified with RGD peptide (DS 10, 10 RGD
peptides per alginate chain), as previously described (Cezar C A,
et al. (2014) Adv. Healthcare Mater. 3, 1869-1876). While
non-degradable alginate was used to simplify the system, oxidized
alginate can be used in the future to create ferrogels with
controlled degradation. In this case, optimization of iron oxide
nanoparticle size, shape, and surface charge may be necessary to
ensure particle absorption by the lymphatic capillary system and
clearance from the body. Prior to implantation, biphasic ferrogel
scaffolds were hydrated with 100 .mu.L PBS. Biphasic ferrogel
scaffolds were then placed subcutaneously on the tibialis anterior
muscle and stimulated for 5 min at 1 Hz every 12 hours by
approaching and retracting a permanent magnet with a surface field
of 6510 Gauss (K&J Magnetics, DXOZO). The ferrogel implant and
magnet were orientated so that magnetic stimulation resulted in
compression of the ferrogel against the injured muscle. Following
retrieval at 3 days and 2 weeks, scaffolds were fixed in 10%
neutral-buffered neutral buffered formalin overnight. Ferrogels
were then paraffin embedded, sectioned at 7 .mu.m thickness, and
stained with hematoxylin and eosin at the Harvard Rodent
Histopathology Core. For pressure profile generation, an Instron
3342 single column apparatus (10 N load cell) configured for
tensile testing was used. A custom adapter that allowed for
stimulation of the ferrogel far from the load cell was attached to
the top tensile grip, and the ferrogel was cyclically stimulated
with a permanent magnet.
Pressure Cuff Stimulation
For mechanical stimulation using the balloon pressure cuff, a
spherical low durometer urethane balloon (10-15 mm diameter
10000000FA, Vention medical) was attached to flexible silicone
tubing (1.78 mm outer diameter, 1.1 mm inner diameter, McMaster
Carr) and bonded at each neck with UV cure adhesive (Loctite 3943,
Henkel). The output from two solenoid valves (TE miniature
switching solenoid valves, sensor technics) was joined and
connected to the balloon. Valve inlets were connected to a
pneumatic air pump (pneumatic pump M0019057, Parker electronics) at
the pressure and vacuum outlets respectively. The valves were wired
to 2-channel relay board with two SRD-05 VDC-SL-C relays that were
controlled by an Arduino Uno microcontroller developer board. The
microcontroller was programmed to sequentially open the valves for
500 ms every second so that the balloon would be pressurized for
500 ms and then evacuated for 500 ms at a 1 Hz frequency. The
balloon was placed inside a polycarbonate cylindrical cuff made
from a 20 ml syringe body trimmed to length with the plunger
removed (20 mm outer diameter, 40 mm length, adjustable wall
thickness from 2-4 mm using polycarbonate cylindrical liners). For
pressure cuff stimulation, the mouse leg was placed in the cuff and
the balloon was fixed directly above the tibialis anterior muscle
outside the skin. The balloon was then inflated and deflated for 5
min at 1 Hz every 12 h. For pressure profile generation, an Instron
3342 single column apparatus (10 N load cell) configured for
compression testing was used. The balloon was removed from the
cylindrical cuff and cyclically inflated and deflated between two
stationary compression platens separated by a distance equal to the
diameter of the deflated balloon.
Histologic Assessment of Skeletal Muscle
Mice were sacrificed and hindlimb muscle tissues (n=10) were
processed for histologic analyses. Tibialis anterior muscles were
fixed in 10% neutral buffered formalin overnight, paraffin
embedded, and sectioned at 7 .mu.m thickness. Sections stained with
H&E were used for quantification of mean fiber diameter,
inflammatory infiltrate, and fibers with centrally located nuclei.
All histologic analyses performed on H&E stained sections were
performed in a blinded fashion. M1 macrophages were identified with
immunostaining for mouse CCR7 (Abcam ab32527; Invitrogen A-11035).
Vascular endothelial cells were identified by immunostaining for
mouse CD31 (Abcam ab28364; Invitrogen A-11035). Interstitial
fibrosis was assessed in picosirius stained sections imaged with
polarized light. Quantification of CCR7 and CD31 immunostaining was
performed using ImageJ.
Muscle Function Testing
Intact tibialis anterior muscles were dissected (n=5/condition),
mounted vertically midway between two cylindrical parallel steel
wire electrodes (1.6 mm diameter, 21 mm long) attached by their
tendons to microclips connected to a force transducer (FORT 25,
WPII) and bathed in a physiologic saline solution in a chamber with
continuously bubbled oxygen at 37.degree. C. Muscle length was
adjusted to a physiological relevant length. A wave pulse was
initiated using a custom-written LabVIEW program and delivered to
the stimulation electrodes via a purpose-built power amplifier (QSC
USA 1310). Contractions were evoked every 5 min. Tetani was evoked
at 250-300 Hz and 25-30 V, with a constant pulse width of 2 ms and
a train duration of 1 s. Peak tetanic force was determined as the
difference between the maximum force during contraction and the
baseline level. Forces were then normalized to muscle wet
weight.
Oxygen Probe
An OxyLab pO2 instrument and implantable optical probe (Oxford
Optronix, Oxford, UK) were used to measure rapid temporal changes
in intramuscular dissolved oxygen and temperature during biphasic
ferrogel stimulation. Briefly, the probe was inserted using a 16 g
needle catheter, and the tip was carefully placed in the midbelly
region of the tibialis anterior muscle. A biphasic ferrogel was
then implanted subcutaneously above the muscle containing the probe
as before (Cezar C A, et al. (2014) Adv. Healthcare Mater. 3,
1869-1876), and the incision was closed around the probe. Oxygen
and temperature readings were recorded continuously before, during,
and after biphasic ferrogel stimulation at 1 second intervals.
Statistical Analyses
All statistical comparisons were performed using ANOVA with
Bonferroni's post-hoc test and a two-tailed unpaired Student's
t-test with Welch's correction and analyzed using INSTAT 3.1a
(GraphPad Software, Inc., San Diego, Calif., USA) software.
Differences between conditions were considered significant if
p<0.05.
Example 1. Experimental Design and Muscle Stimulation Profiles
The tibialis anterior muscle of each C57BL6/J mouse was subjected
to a severe dual injury involving an intramuscular injection of
notexin followed by induction of hindlimb ischemia six days later,
as previously described (Borselli C et al., (2011) Biomaterials
32(34):8905-8914). Complete loss of locomotion of the injured
hindlimb was observed immediately following induction of ischemia.
Following ischemic surgery, the injured muscle was treated with a
subcutaneously implanted biphasic ferrogel, subsequently stimulated
at 1 Hz for 5 min every 12 hrs noninvasively using a permanent
magnet (FIG. 1A). Control conditions included a pressure cuff
stimulated at 1 Hz for 5 min every 12 hours, a biphasic ferrogel
without stimulation, magnetic field only, and no treatment. The
biphasic ferrogel provides stimulation directly to the muscle. The
pressure cuff externally compresses the muscle through the skin.
Both stimulation of the biphasic ferrogel and the pressure cuff led
to uniform cyclic compressions on the injured muscle. Importantly,
biphasic ferrogels exhibited fatigue resistance as Young's modulus
and toughness changed minimally throughout the 2 week study (FIGS.
7A and 7B). Stimulated biphasic ferrogels were able to exert a peak
pressure of 1.2 kPa while the pressure cuff was able to exert a
slightly larger peak pressure of 2.0 kPa. The kinetics of the
compressions varied between the biphasic ferrogel and the pressure
cuff, with the biphasic ferrogels exhibiting more gradual changes
in pressure while the pressure cuff demonstrated more rapid changes
in pressure (FIGS. 1B and 1C).
Example 2. Host Response to Ferrogel Implant
Biphasic ferrogels were histologically examined 3 days and 2 weeks
following implantation in order to determine the host response to
the implants. Upon retrieval, all scaffolds were found localized at
the initial site of implantation. Initial orientation of the gel
relative to the skin and injured muscle tissue remained unchanged
throughout the study. Additionally, no significant differences in
gel thickness were observed between stimulated and non-stimulated
ferrogels. At 3 days, both stimulated and non-stimulated biphasic
ferrogels remained largely acellular and no fibrous capsule was
seen surrounding the implants. At 2 weeks, a fibrous capsule
surrounding all biphasic ferrogel implants was observed (FIG. 2A).
Non-stimulated biphasic ferrogels were surrounded with a capsule of
.about.120 .mu.m thickness while stimulated biphasic ferrogels were
surrounded with a significantly thinner capsule of .about.75 .mu.m
thickness (FIG. 2B).
Example 3. Markers of Muscle Regeneration: Centrally Located Nuclei
and Muscle Fiber Size
In order to determine the histological quality of muscle
regeneration, several markers of muscle regeneration were examined.
Areas of active muscle regeneration were present in all treatment
conditions as indicated by muscle fibers containing centrally
located nuclei (Hawke T J & Garry D J (2001) J Appl Physiol
(1985) 91(2):534-551). Although no statistically significant
differences were observed at 2 weeks, greater than 40% of fibers
contained centrally located nuclei in all conditions except the no
treatment control (30%) (FIGS. 3A and 3B). Mean muscle fiber size,
as measured by cross-sectional area, remained fairly constant
(.about.100 .mu.m.sup.2) in all tested conditions 3 days
post-injury, as expected. However, at 2 weeks, mean muscle fiber
size was generally greater in muscles treated with stimulated
biphasic ferrogels (205 .mu.m.sup.2) when compared to no treatment
controls (130 .mu.m.sup.2) (FIGS. 3A and 3C). Interestingly, a
significant increase in mean muscle fiber size from 3 days to 2
weeks was only seen in muscles treated with stimulated biphasic
ferrogels.
Example 4. Inflammation: Interstitial Fibrosis, Inflammatory
Infiltrate, and Macrophage Presence
The influence of cyclic mechanical compressions on inflammation and
fibrosis was next examined using histologic sections. The extent of
muscle fibrosis was assessed by visualizing picosirius red stained
collagen I and III under polarized light. Interestingly, at 2
weeks, stimulated biphasic ferrogels showed significantly less
interstitial fibrosis when compared to no treatment controls (FIGS.
4B and 4E). In contrast, all other treatment conditions remained
statistically equivalent at this time point. In addition,
quantification of the inflammatory infiltrate followed a similar
trend. Unlike all other conditions, a significantly lower number of
muscle infiltrating inflammatory cells were observed in stimulated
biphasic ferrogel conditions when compared to no treatment controls
(FIGS. 4A and 4D). Finally, stimulation of injured muscles by
biphasic ferrogels and pressure cuff controls significantly reduced
M1 macrophage infiltration, as measured by CCR7 staining (FIGS. 4C
and 4F).
Example 5. Angiogenesis, Hindlimb Perfusion, and Oxygen
The ability of cyclic mechanical compressions to promote hindlimb
reperfusion and angiogenesis was examined next. Induction of
hindlimb ischemia led to a dramatic decrease in perfusion relative
to the contralateral control limb, from 100% to .about.30%
immediately following surgery in all treatment groups (FIG. 5A), as
expected. At 3 days, perfusion increased to .about.40% in all
conditions. A difference between the stimulated and non-stimulated
biphasic ferrogel conditions appeared at day 9, but did not persist
to day 14. A significant increase in perfusion of the muscle tissue
was observed upon treatment of the pressure cuff (FIG. 5D).
Differences in the average capillary density in muscle sections
between the various conditions, as measured by immunostaining for
the endothelial cell marker CD31, were not observed (FIG. 5B). The
oxygen concentration within the muscle remained at a fairly
constant baseline level of .about.20 mmHg before biphasic ferrogel
stimulation (FIG. 5C). Upon stimulation, however, intramuscular
oxygen concentration rapidly increased and remained elevated until
stimulation ceased. Upon cessation of ferrogel stimulation, the
oxygen concentration rapidly returned to the previous baseline
levels.
Example 6. Muscle Function: Contraction Force
To assess the functional quality of muscle regeneration, the
contractile force of each injured and contralateral control muscle
was measured. At 2 weeks, injured muscles treated with stimulated
biphasic ferrogels and pressure cuffs showed significant increases
in specific peak tetanic force, 2.6 and 2.2 fold over no treatment
controls, respectively (FIG. 6). Stimulated biphasic ferrogels also
showed a significant increase in specific peak tetanic force over
the magnetic field only control (1.9 fold). In contrast, no
significant difference in specific peak tetanic force was observed
between the no treatment, magnetic field only, and non-stimulated
biphasic ferrogel conditions.
To understand whether cyclic compressive stimulation improves
muscle regeneration in a temporal manner, pressure-cuff mediated
mechanical stimulation was applied on the injured taibialis
anterior muscle for different durations (3, 7, and 14 days) at 1 Hz
for 5 minutes every 12 hours with a peak pressure of 2.0 kPa. As
shown in FIG. 9, the contractile force of injured muscle was
progressively enhanced during the 14-day course of treatments. The
average contractile force of the muscle treated with mechanical
stimulation for 14 days was significantly higher than their control
counterpart (no stimulation).
Example 7. Effect of Cyclic Mechanical Compression on Cytokine
Levels
The effect of cyclic mechanical compressions on modulating the
levels of cytokines in injured tissue was examined next. Briefly,
113 cytokines were screened from lysates of the tibialis anterior
muscle treated with and without mechanical stimulation for 7 days
after ischemic injury. As shown in FIG. 10, around 70 out of 113
cytokines were found to have a lower level of expression (top) in
the muscle treated with mechanical stimulation by varying degrees
as compared to their control counterparts (from 1.1 to 2.7 times).
In addition, the molecules most significantly decreased in the
muscle treated with mechanical compressions are closely associated
with pro-inflammatory responses such as myeloperoxidase, neutrophil
gelatinase-associated lipocalin, interleukin-17A and interleukin-6.
Only a few cytokines were detected to have a higher level of
expression (bottom) in the muscle tissue treated with mechanical
compressions (up to 1.2 times). These data indicate that mechanical
compressions indeed can modulate the levels of cytokines in injured
tissue.
Subsequently, the effect of cyclic mechanical compression on
intramuscular convection was assessed. Briefly, fluorescently
labeled dextran (40 kDa) was injected into the tibialis anterior
muscle and the change in fluorescent intensity of the injected
dextran was monitored before and after mechanical compression by In
Vivo Imaging Instruments. As shown in FIG. 11, the fluorescent
intensity of the dextran significantly increased in the muscle
treated with mechanical stimulation relative to their control group
(no stimulation). This might be because cyclic compression caused a
change in intramuscular convection, for example, by expelling the
intramuscularly injected dextran out of the muscle toward the skin,
and consequently the signals from molecules close to the skin might
contribute to the enhanced fluorescent intensities.
Overall, these findings indicate that cyclic mechanical
compressions can modulate the levels of various cytokines in the
injured muscle tissue, which can potentially influence the
regenerative processes of injured muscle. Moreover, this change in
the cytokine levels might be due to the altered intramuscular
convection by cyclic compression.
DISCUSSION
The present invention demonstrated that direct mechanical
stimulation can enhance the regeneration of severely damaged
skeletal muscle, obviating the need for exogenous growth factors or
cells. These studies were carried out using a murine model of
severe muscle injury involving both myotoxin-induced direct muscle
damage and hind limb ischemia. This model leads to substantial
necrosis of the muscle, fibrosis and loss of significant
contractile function (Borselli C et al., (2011) Biomaterials
32(34):8905-8914), mimicking severe injuries in humans.
Actuation of biologic-free ferrogels resulted in mechanical
compressions that affected the host inflammatory response towards
the gel and led to a reduction in fibrous capsule thickness
following 2 weeks of implantation. Strikingly, mechanical
stimulation led to a significant reduction in fibrosis and
inflammation of the injured muscle, demonstrating a potential
immunomodulatory role for ferrogel-driven cyclic compressions. As
assessed histologically, severe muscle injury resulting from
myotoxin injection and hindlimb ischemia led to subsequent active
muscle regeneration that was enhanced by stimulated biphasic
ferrogels. In addition, mechanical stimulation led to a temporary
increase in oxygen concentration at the site of injury.
Biologic-free ferrogel and pressure cuff driven mechanical
compressions led to enhanced muscle regeneration and muscle
function when compared to no treatment controls, demonstrating the
therapeutic potential of these mechanical interventions.
Actuation of both the biphasic ferrogel and the pressure cuff
results in uniform cyclic compressions of the severely injured
muscle tissue. The pressure cuff was roughly tuned so that the peak
pressure values achieved by each system were comparable. The peak
pressure value achieved by the pressure cuff remained slightly
larger than that achieved by the biphasic ferrogel, and the
kinetics of the pressure profiles also varied. Both the biphasic
ferrogel and pressure cuff systems were able to apply a force
(normalized to tibialis anterior wet weight) of .about.2 N/g, a
value similar to that used previously for massage-like compressive
loading of rabbit tibialis anterior muscles injured with eccentric
exercise (Haas C, et al. (2013) Br. J. Sports Med. 47(2):83-88).
Stimulation parameters may be chosen to yield mechanical
compressions that approximate those achieved with massage. In
addition, the frequency, amplitude, and duration of the
stimulations may be optimized for the tissue injury being
addressed.
Biphasic ferrogel stimulation leads to a reduction in fibrous
capsule thickness and inflammatory cells present in the surrounding
muscle following 2 weeks of implantation, and this may relate to
expulsion of inflammatory cells due to cyclic compression of the
ferrogels. It is possible that invading cells near the scaffold
edges were expelled from the ferrogel system upon stimulation, due
to fluid convection resulting from large gel deformations, leading
to an overall diminished cell presence within the scaffold. The
decrease in M1 macrophage presence with ferrogel stimulation
further suggests a potent immunomodulatory role for cyclic
mechanical compressions. Taken together, these studies provide
evidence that cyclic compressions, e.g., ferrogel-driven cyclic
compressions and pressure cuff-driven compressions, may be useful
to alleviate inflammation in certain tissue, e.g., muscle injuries.
Further, this ability to inhibit fibrous capsule formation with
cyclic compressions has potential utility for implantable drug
delivery devices and sensors that require unobstructed diffusion
around the implant for proper function.
Severe muscle injury resulting from myotoxin injection and hindlimb
ischemia leads to active muscle regeneration that can be enhanced
with stimulated biphasic ferrogels. While centrally located nuclei
were observed in all conditions, significant increases in the mean
muscle fiber size of regenerating fibers over time were only
observed in muscles treated with stimulated biphasic ferrogels.
Past reports suggest increased mean fiber diameter is indicative of
tissues that have progressed further in the regenerative process
(Borselli C, et al. (2010) Proc. Natl. Acad. Sci. U.S.A.
107(8):3287-3292; Wang L, et al. (2014) Mol. Ther. 22(8),
1441-1449).
Mean muscle fiber size values remained remarkably consistent among
all treatment groups at this time point. At 2 weeks, pressure cuff
controls exhibited a smaller mean fiber size than stimulated
ferrogels suggesting that the ferrogel-driven cyclic mechanical
compressions provide an additional, likely convection-based
benefit. Specifically, enhanced fluid transportation around the
implant site may accelerate immune cell and/or metabolic waste
product removal. Interestingly, ferrogel-driven mechanical
stimulation produces a therapeutic effect on muscle fiber size of
the same order of magnitude as that achieved by previous approaches
that delivered cells and drugs to severely injured muscle (Borselli
C, et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107(8):3287-3292;
Wang L, et al. (2014) Mol. Ther. 22(8), 1441-1449).
Cyclic mechanical compressions do not lead to an enhancement in
hindlimb reperfusion and angiogenesis, but may instead lead to
temporary increases in convection through the tissue or blood flow
to the injured limb. While small differences in perfusion were
present 9 days post-injury, they did not persist to the end of the
study, suggesting that growth factor or cell support may be
required to maintain any increases in perfusion that appear due to
cyclic mechanical compressions alone. Strikingly, although
steady-state hindlimb perfusion and capillary density were not
significantly affected, oxygen probe experiments demonstrated an
increase in oxygen concentration during the time in which biphasic
ferrogel-driven cyclic mechanical compressions were being generated
in the muscle. Likely, enhanced intramuscular convection driven by
tissue compressions may have led to increased oxygen levels and
expedited removal of metabolic byproducts that inhibit
regeneration. Alternatively, cyclic compressions may increase
oxygen concentration by locally and temporarily increasing blood
flow to the injured muscle. Strikingly, biphasic ferrogel and
pressure cuff driven cyclic mechanical compressions of injured
muscle led to significant functional muscle regeneration. Following
2 weeks of treatment, biphasic ferrogel and pressure cuff treatment
conditions showed 2.6- and 2.2-fold increases in peak tetanic force
over no treatment conditions, respectively. While additional
biomaterial-based approaches have induced similar improvements in
muscle function through the co-delivery of myogenic bioagents with
endothelial cells to enhance vascularization (5.5 fold increase in
active stress over blank scaffold) or neural stem cells to promote
innervation of muscle constructs (2.0 fold increase in maximum
tetanic force over nerve-deficient control) (Koffler J, et al.
(2011) Proc. Natl. Acad. Sci. U.S.A. 108(36):14789-14794; Shandalov
Y, et al. (2014) Proc. Natl. Acad. Sci. U.S.A. 111(16):6010-6015;
Morimoto Yi et al., (2013) Biomaterials 34(37):9413-9419; Larkin L
M, et al., (2006) In Vitro Cell. Dev. Biol. Anim. 42(3-4):75-82;
Rowley J A et al, (1999) Biomaterials 20(1):45-53; Chen R R, et al.
(2007) FASEB J. 21(14):3896-3903), no bioagents were delivered from
the scaffolds in this study. The current study is the first report
of functional muscle regeneration due to cyclic mechanical
compressions in a severe model of muscle damage.
Strikingly, the results of these studies indicate a ferrogel
scaffold and pressure cuff can be used to mechanically stimulate
and regenerate severely injured muscle tissue without the use of
growth factors or cells. The demonstration of functional muscle
regeneration with a biologic-free material system may offer a
simple yet effective alternative to cell-based therapies when
treating certain types of muscle injuries. Further, incorporation
of cyclic mechanical compressions into existing drug and cell
delivery systems could potentially lead to a new combinatorial
therapies that generate enhanced regenerative outcomes. While this
study focuses on the repair of skeletal muscle, bioagent-free
devices, such as ferrogels and pressure cuffs, and the concept of
mechanically driven regeneration are expected to find broad utility
and can likely be applied to other tissues and diseases.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. such
equivalents are intended to be encompassed by the following
claims.
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